Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles

TRAIL has received considerable attention as a potential anti-cancer agent due to its specific ability to target tumors. However, recombinant TRAIL has several limitations, such as, its short biological half-life, its inherent instability, and its potential hepatotoxicity. In this study, we developed a sustained release nanoparticle formulation of TRAIL and investigated its therapeutic effects in tumor-bearing mice. TRAIL-loaded nanoparticles (NPs) were prepared by mixing PEGylated heparin (PEG-HE), poly-L-lysine (PLL), and TRAIL. NPs prepared by the ionic interaction between polymer and TRAIL showed uniform spherical structures of diameter 213.3 ± 9.7 nm and a surface charge of 5.33 ± 1.2 mV. An in vitro study of the bioactivity of TRAIL in NPs showed that TRAIL-loaded PEG-HE/PLL NPs (TRAIL-PEG-NPs) were slightly less cytotoxic than TRAIL in vitro. To investigate pharmacokinetic parameters, TRAIL and TRAIL-PEG-NPs were intravenously injected into SD rats. The PEG-NP-based formulation demonstrated a 28.3 fold greater half-life than TRAIL alone. To evaluate the anti-tumor effect, TRAIL, TRAIL-loaded HE/PLL NPs (TRAIL-NPs), and TRAIL-PEG-NPs were intravenously injected into HCT-116 tumor-bearing BALB/c athymic mice. The TRAIL-PEG-NP formulation efficiently suppressed tumor growth (>70%), and histological findings confirmed that NPs induced significant tumor cell apoptosis without inducing liver toxicity. The PEG-exposed NP fabrication method applied in this study could be widely applied to protein and peptide delivery systems.     

Anti-tumor activity of paclitaxel through dual-targeting carrier of cyclic RGD and transferrin conjugated hyperbranched copolymer nanoparticles

Targeted delivery strategies are becoming increasingly important. Herein, a novel hyperbranched amphiphilic poly[(amine-ester)-co-(d,l-lactide)]/1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine copolymer (HPAE-co-PLA/DPPE) with RGD peptide (cRGDfK) and transferrin (Tf) on the periphery was synthesized and used to prepare paclitaxel-loaded nanoparticles (NPs) for dual-targeting chemotherapy. These NPs show satisfactory size distribution, high encapsulated efficiency and a pH-dependent release profile. The intrinsic fluorescence of the hyperbranched copolymer renders the detection and tracking of NPs in vitro and in vivo conveniently. In vitro cytotoxicity studies proved that the presence of cRGDfK enhanced the cytotoxic efficiency by 10 folds in α_νβ₃ integrin over-expressed human umbilical vein endothelial cells, while Tf improved cytotoxicity by 2 folds in Tf receptor over-expressed human cervical carcinoma cells. The drug-loaded NPs can be efficiently transported into the vascular endothelial cells and the target tumor cells. These results indicate that the cRGDfK and Tf decorated HPAE-co-PLA/DPPE could deliver chemotherapies specifically inside the cell via receptor-mediated endocytosis with greater efficacy. Therefore, such a fluorescent nanocarrier prepared from non-cytotoxic and biodegradable polymers is promising for drug delivery in tumor therapy.     

DNA methyltransferase 1 is essential for initiation of the colon cancers

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.     

Phenotype of NK Cells and Cytotoxic/Apoptotic Mediators Expression in Ectopic Pregnancy

Citation Laskarin G, Redzovic A, Vukelic P, Veljkovic D, Gulic T, Haller H, Rukavina D. Phenotype of NK cells and cytotoxic/apoptotic mediators expression in ectopic pregnancy. Am J Reprod Immunol 2010 Problem The expression of cytotoxic/apoptotic mediators and the phenotype characteristics of uterine NK cells (uNK) in tubal ectopic pregnancy (EP) were investigated. Method of study Samples of uterine decidua and tubal mucosa as well as peripheral blood (PB) of the same women with EP were used for phenotype characterization of NK cells and detection of cytotoxic/apoptotic mediators and IL‐15. Results In tubal mucosa, perforin, FasL, granulysin and IL‐15 were almost completely absent, but they were present in normal and EP uterine deciduas. TRAIL was present on trophoblast and tubal mucosa, contrary to its lack in normal and EP uterine decidua. CD16^?CD56^dim NK cells, mostly CD94^? and NKG2A^?, predominate in tubal mucosa, whereas CD16^?CD56^bright NK cells, predominantly CD94⁺ and NKG2A⁺ prevail in EP uterine decidua. NK cells at the EP implantation site express lower percentages of perforin and granulysin, but they express a higher percentage of TRAIL than do EP uterine decidual and PB NK cells. Lower percentage of TNF‐α‐expressing and IL‐4‐expressing NK cells were found at the implantation site compared to EP uterine decidua. Conclusions Authentic uNK cell population seems to be insufficient to restrict trophoblast invasion because of low expression of cytotoxic/apoptotic mediators.     

阿司匹林增强TRAIL诱导的HepG-2细胞凋亡

目的:研究阿司匹林能否增强肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导的HepG-2细胞凋亡,并初步探讨其作用机制。方法:HepG-2细胞放入含15%胎牛血清的DMEM培养液中培养,改良苯酚-品红染色观察细胞形态变化,MTT法检测细胞增殖程度,TUNEL法检测细胞凋亡指数,RT-PCR检测细胞中survivinmRNA的表达水平,FCM检测细胞凋亡率和细胞周期。结果:不同浓度的阿司匹林联合TRAIL实验组细胞增殖抑制率明显高于空白对照组及TRAIL和阿司匹林单独用药组,且细胞凋亡指数也明显提高,凋亡率与阿司匹林的浓度呈现剂量依赖关系,联合用药组凋亡相关基因survivin的表达与空白对照组和TRAIL和阿司匹林单用组相比明显下调。结论:阿司匹林能够增强TRAIL诱导的HepG-2细胞凋亡,其作用机制可能与survivin基因的表达下调有关。     

Targeted Delivery of Doxorubicin Using a Colorectal Cancer‐Specific ssDNA Aptamer

Targeted drug delivery is particularly important in cancer treatment because many antitumor drugs are nonspecific and highly toxic to both cancerous and normal cells. The L33 aptamer is a single‐stranded DNA (ssDNA) sequence that has the ability to recognize human colorectal cancer (CRC) cell line HCT116 specifically. In this study, we demonstrated that the L33 aptamer can selectively internalize into target HCT116 cells via receptor‐mediated endocytosis. Based on this finding, we developed an aptamer‐based drug delivery system using L33 as the carrier of the antitumor drug doxorubicin (Dox). The L33‐Dox complex exhibited specific and high affinity (K_d = 14.3 ± 2.2 nM) binding to HCT116 cells. The results of cytotoxicity assays revealed that the L33‐Dox complex was capable of selectively delivering the drug to the target HCT116 cells and lowered the toxicity for nontarget CL187 cells. These findings indicate that the aptamer‐based targeted drug delivery system has the potential to be used in clinical settings and may overcome drug resistance to a certain extent because high drug dosages can be directed toward target cells. Anat Rec, 297:2280–2288, 2014. © 2014 Wiley Periodicals, Inc.     

Haematopoietic stem cells and progenitors of functional T-lymphocytes in the bone marrow of `nude'' mice

The stem cell potential of thymusless nu/nu mice measured in terms of CFU ratio and the capacity to reprieve lethally irradiated mice was similar to that of nu/+ and CBA mice. The thymus of nu/nu→T6T6 rediation chimaeras was repopulated by donor cells. Proliferation of donor cells, presumed to be T-lymphocytes, was observed in regional lymph nodes 3 days after sensitization with oxazolone.

T6T6 thymus grafts implanted subcutaneously in nu/nu mice restored the response to C57BL skin grafts. The mitotic figures in the thymus implant 30 and more days after grafting were almost exclusively of host karyotype. The high proportion of host cells dividing in the lymph nodes of thymus-grafted nu/nu mice suggested that T-cells derived originally from nu/nu bone marrow were present. Allogeneic thymus grafts partially restored the response of nu/nu mice to oxazolone.

It is concluded that the failure of thymic development in nu/nu mice is not due to an absence of thymocyte precursors in the bone marrow or to anything in the general physiological environment which prevents their differentiation to thymocytes and T-lymphocytes. It is presumably the result of defective embryonic development of the epithelial rudiment.

     

Four-arm PEG cross-linked hyaluronic acid hydrogels containing PEGylated apoptotic TRAIL protein for treating pancreatic cancer

Four-arm polyethylene glycol (PEG) cross-linked hyaluronic acid (HA) hydrogels containing PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) were fabricated, and their antitumor effects were evaluated in pancreatic cell (Mia Paca-2)-xenografted mice. HA was conjugated with 4-arm PEG_10k-amine (a cross-linker) at ratios of 100:1 and 100:2 using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a cross-linker, and TRAIL or PEG-TRAIL was incorporated into these HA hydrogels. HA hydrogels at a 100:1 ratio were prepared in good yields (>88%), were moderately stiff, and gradually released PEG-TRAIL over ∼14 days in vitro and over ∼7 days in vivo (as determined by high-pressure liquid chromatography and infrared imaging). The released PEG-TRAIL was found to have obvious apoptotic activity in Mia Paca-2 cells. PEG-TRAIL HA hydrogels displayed remarkably more antitumor efficacy than TRAIL HA hydrogels in Mia Paca-2 cell-xenografted mice in terms of tumor volumes (size) and weights (453.2 mm³ and 1.03 g vs. 867.5 mm³ and 1.86 g). Furthermore, this improved antitumor efficacy was found to be due to the apoptotic activity of PEG-TRAIL in vivo (determined by a TUNEL assay) despite its substantially lower cytotoxicity than native TRAIL (IC₅₀ values: 71.8 and 202.5 ng ml⁻¹, respectively). This overall enhanced antitumor effect of PEG-TRAIL HA hydrogels appeared to be due to the increased stability of PEGylated TRAIL in HA hydrogels. These findings indicate that this HA hydrogel system combined with PEG-TRAIL should be considered a potential candidate for the treatment of pancreatic cancer.     

Cellular apoptosis susceptibility (chromosome segregation 1‐like,CSE1L) gene is a key regulator of apoptosis,migration and invasion in colorectal cancer

Cellular apoptosis susceptibility (chromosome segregation 1‐like, CSE1L) gene maps to chromosomal region 20q13.13, a region frequently amplified in solid tumours. In this study, we investigated the roles played by CSE1L in colorectal cancer by examining CSE1L expression and clinico‐pathological parameters in colorectal cancer and investigating the effect of CSE1L on the viability, adhesion and migration of colorectal cancer cells. RT‐PCR showed that CSE1L mRNA was over‐expressed in colorectal cancer. CSE1L depletion by knock‐down with CSE1L‐specific siRNA significantly reduced viability in HCT116 cells (p = 0.004) and SW480 cells (p = 0.003) whilst significantly increasing the proportion of apoptotic HCT116 cells (p < 0.001) and SW480 cells (p < 0.001). Furthermore, CSE1L depletion significantly reduced the adhesive capacity of HCT116 (p = 0.003) and SW480 cells (p = 0.004). Analysis by qRT‐PCR following CSE1L siRNA treatment of HCT116 and SW480 cells showed significant modulation of key apoptotic (p53, p73 and BAK) and adhesive (E‐cadherin, Ep‐CAM and ICAM‐1) molecules. Immunohistochemistry of a colorectal cancer tissue microarray showed that CSE1L had a significantly increased level in colorectal cancer compared to normal colorectal epithelium (p < 0.001). There were significant decreases in both nuclear (p = 0.006) and cytoplasmic (p = 0.003) staining of CSE1L in tumours with lymph node metastasis (stage 3 tumours) compared with lymph node‐negative tumours (stage 1 and 2 tumours). In lymph node‐negative patients, poor survival was associated with increased CSE1L cytoplasmic expression (p = 0.042). These results indicate that CSE1L is associated with viability and apoptosis, cellular adhesion and invasion, thus implicating CSE1L in the progression of colorectal cancer. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

Antifungal activity of silver nanoparticles against Candida spp.

The antifungal activity of the silver nanoparticles (NPs) prepared by the modified Tollens process was evaluated for pathogenic Candida spp. by means of the determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and the time-dependency of yeasts growth inhibition. Simultaneously the cytotoxicity of the silver NPs to human fibroblasts was determined. The silver NPs exhibited inhibitory effect against the tested yeasts at the concentration as low as 0.21 mg/L of Ag. The inhibitory effect of silver NPs was enhanced through their stabilization and the lowest MIC equal to 0.05 mg/L was determined for silver NPs stabilized by sodium dodecyl sulfate against Candida albicans II. The obtained MICs of the silver NPs and especially of the stabilized silver NPs were comparable and in some cases even better than MICs of the conventional antifungal agents determined by E-test. The silver NPs effectively inhibited the growth of the tested yeasts at the concentrations below their cytotoxic limit against the tested human fibroblasts determined at a concentration equal to 30 mg/L of Ag. In contrast, ionic silver inhibited the growth of the tested yeasts at the concentrations comparable to the cytotoxic level (approx. 1 mg/L) of ionic silver against the tested human fibroblasts.     

Preclinical study of treatment response in HCT-116 cells and xenografts with (1) H-decoupled (31) P MRS

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN‐38 have been shown to induce G₂/M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT‐116). Subsequent treatment of these G₂/M‐arrested cells with the cyclin‐dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT‐116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton (¹H)‐decoupled phosphorus (³¹P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT‐116 xenografts following treatment, which were evidenced within twenty‐four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT‐116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN‐38 alone underwent 83 ± 5% G₂/M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN‐38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo ¹H‐decoupled ³¹P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies. Copyright © 2011 John Wiley & Sons, Ltd.     

慢病毒介导的RNA干扰沉默Slug基因对结肠癌HCT116细胞增殖和凋亡的影响

目的:探讨利用RNA干扰(RNA interference,RNAi)技术沉默Slug基因,观察对结肠癌HCT116细胞增殖和周期的影响。方法:构建Slug基因特异性siRNA慢病毒载体,感染结肠癌HCT116细胞,设立空白对照组、阴性对照组及SlugsiRNA三组,应用Real-time PCR和Western blot方法分别从基因和蛋白质水平检测各组干扰质粒对Slug基因的干扰效果,MTT法检测Slug基因在siRNA作用下的细胞增殖率,流式细胞仪检测细胞凋亡周期变化情况。结果:转染Slug siRNA后,结肠癌HCT116细胞中Slug基因mRNA和蛋白表达明显受到抑制(P<0.05);MTT检测,干扰组细胞增殖水平明显低于阴性对照组;流式细胞仪检测细胞G1期细胞百分比(52.3±0.6)高于阴性对照组(45.1±0.3,P<0.05)。结论:Slug siRNA能明显下调靶基因Slug的表达,在体外可抑制结肠癌HCT116细胞的生长并促进其凋亡。     

The preparation and characterization of folate-conjugated human serum albumin magnetic cisplatin nanoparticles

Objective

Nanoparticles are becoming an important method of targeted drug delivery. To evaluate the importance of folate-conjugated human serum albumin (HSA) magnetic nanoparticles (Folate-CDDP/HSA MNP), we prepared drug-loaded Folate-CDDP/HSA MNPs and characterized their features.

Methods

First, folate was conjugated with HSA under the effect of a condensing agent, and the conjugating rate was evaluated by a colorimetric method using 2, 4, 6 - trinitrobenzene sulfonic acid. Second, under N₂ gas, Fe₃O₄ magnetic nanomaterials were prepared and characterized by using transmission electron microscopy (TEM), SEM-EDS and X-ray diffraction (XRD). Finally, Folate-CDDP/HSA MNP was prepared by using a solvent evaporation technique. TEM was used to observe particle morphology. The particle size and distribution of the prepared complexes were determined by a Laser particle size analyzer. Drug loading volume and drug release were investigated by a high performance liquid chromatography method (HPLC) in vitro.

Results

We successfully prepared folate-conjugated HSA and its conjugating rate was 27.26 µg/mg. Under TEM, Fe₃O₄ magnetic nanoparticles were highly electron density and had an even size distribution in the range of 10-20 nm. It was confirmed by SEM-EDS and XRD that Fe₃O₄ magnetic nanoparticles had been successfully prepared. Under TEM, drug-loaded magnetic nanoparticles were observed, which had a round shape, similar uniform size and smooth surface. Their average size was 79 nm which was determined by laser scattering, and they exhibited magnetic responsiveness. Encapsulation efficiency was 89.75% and effective drug loading was calculated to be 15.25%. The release results in vitro showed that the half release time (t½) of cisplatin in cisplatin Solution and Folate-CDDP/HSA MNP was 65 min and 24 h respectively, which indicated that microspheres had an obvious effect of sustained-release.

Conclusion

Folate-CDDP/HSA MNPs were prepared successfully. The preparation process and related characteristics data provided a foundation for further study, including the mechanism of the nanoparticles distribution in vivo and their intake by tumor cells.     

Tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy cooperate to induce apoptosis in mesothelioma cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in certain tumor cells. In addition, TRAIL and chemotherapy can act cooperatively, possibly as a result of chemotherapy-induced increases in expression of a TRAIL receptor, DR5. We used cell lines derived from a highly chemoresistant tumor, malignant mesothelioma, to learn whether TRAIL was effective alone or together with chemotherapy and whether cooperativity depended on increases in DR5 expression. TRAIL (codons 95-285) was expressed in a bacterial expression vector and purified by nickel affinity chromatography. TRAIL alone (25 to 500 ng/ml) had little effect on mesothelioma cells. TRAIL plus chemotherapy (doxorubicin, cis-platinum, etoposide, or gemcitabine) acted cooperatively to induce apoptosis in mesothelioma cells (M28, REN, VAMT, and MS-1). For example, in M28 cells treated for 18 h, apoptosis from TRAIL (100 ng/ml) plus doxorubicin (0.6 microg/ml; 71 +/- 11%) greatly exceeded that from TRAIL alone (21 +/- 8%) or from doxorubicin alone (6 +/- 2%) (means +/- standard deviation; P < 0.03). Mesothelioma cells treated with chemotherapy showed no change in DR5 protein by Western analysis or by immunocytochemistry. TRAIL plus chemotherapy was associated with an increase in mitochondrial cytochrome c release and mitochondrial depolarization. We conclude that TRAIL and chemotherapy act cooperatively to kill mesothelioma cell lines, not by increases in DR5 receptor but in association with mitochondrial amplification of apoptotic signals.     

Core-Shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core–shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.     

核心蛋白聚糖抑制结直肠腺癌细胞系HCT116的体外侵袭

目的探讨常氧和低氧条件下核心蛋白聚糖(DCN)对结直肠腺癌细胞系HCT116体外侵袭的作用及机制。方法 Transwell法检测HCT116细胞体外侵袭能力;Real-time PCR法检测细胞CD133和TIMP-2 mRNA的表达;Western blot检测细胞HIF-1α、CD133和TIMP-2蛋白的表达。结果 1)常氧和低氧环境下,DCN浓度为0、1和3mg/L时HCT116细胞的侵袭数量分别为(241±46)、(168±46)和(51±17)个/视野(P0.01)及(207±61)、(213±64)、(156±54)和(44±17)个/视野(P0.01)。2)在常氧条件下,DCN(3 mg/L)显著上调HCT116细胞TIMP-2mRNA和蛋白的表达(P0.01),而对CD133 mRNA和蛋白的表达无明显作用。3)低氧条件下DCN(3 mg/L)显著下调HCT116细胞HIF-1α/CD133/TIMP-2蛋白的表达(P0.01),而对CD133 mRNA的表达无明显影响。结论DCN抑制结直肠腺癌细胞HCT116体外侵袭能力,在常氧和低氧条件下可能通过不同机制发挥作用。     

Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype

We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LovO, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase δ. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these fouor cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.     

Curcumin induces permanent growth arrest of human colon cancer cells: link between senescence and autophagy

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53−/− cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53−/− HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.