Recombinant adenovirus encoding H-ras ribozyme induces apoptosis in laryngeal cancer cells through caspase- and mitochondria-dependent pathways

Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.     

Theoretical,in Vitro Antiproliferative,and in Silico Molecular Docking and Pharmacokinetics Studies of Heteroleptic Nickel(II) and Copper(II) Complexes of Thiosemicarbazone-Based Ligands and Pefloxacin

Twelve new heteroleptic nickel(II) and copper(II) complexes of the type [M(L^1–6)(Pfx)₂] ( 1 – 12 ), where L^1–6=2-benzylidenehydrazinecarbothioamide (L¹), 2-benzylidene-N-methylhydrazinecarbothioamide (L²), 2-benzylidene-N-phenylhydrazinecarbothioamide (L³), 2-(4-methylbenzylidene)hydrazinecarbothioamide (L⁴), 2-(4-methylbenzylidene)-N-methylhydrazinecarbothioamide (L⁵) and 2-(4-methylbenzylidene)-N-phenylhydrazinecarbothioamide (L⁶), Pfx=pefloxacin and M=Ni(II) or Cu(II) have been synthesised, and their structures were confirmed by different spectral techniques. The spectral data and density functional theory (DFT) calculations supported the bonding of pefloxacin drug molecule via one of the carboxylate oxygen atoms and the pyridone oxygen atom, and the thiosemicarbazone ligand via the imine nitrogen and the thione sulfur atoms with the metal(II) ion, forming distorted octahedral geometry. In vitro antiproliferative activity of the synthesized complexes was evaluated against three human breast cancer (T47D, estrogen negative (MDA-MB-231) and estrogen positive (MCF-7)) as well as non-tumorigenic human breast epithelial (MCF-10a) cell lines, which showed the higher activity for the copper(II) complexes. The interaction of the synthesized complexes with an oncogenic protein H-ras (121 p) was explored by in silico molecular docking studies. Further, in silico pharmacokinetics and ADMET parameters were also analysed to predict the drug-likeness as well as non-toxic and non-carcinogenic behavior, and safe oral administration of the complexes.     

Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells

The present study is designed to investigate the effect of myocardial preconditioning with oxidative stress induced by pyrogallol or H₂O₂, on ischaemia-reperfusion induced myocardial injuiry. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of preconditioning induced by pyrogallol or hydrogen peroxide (H₂O₂) or ischaemia markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. Administration of polymyxin B, a protein kinase C (PKC) inhibitor, during pyrogallol, H₂O₂ or ischaemic preconditioning markedly attenuated the cardioprotective effect of preconditioning produced with oxidative stress or ischaemia. These results suggest that preconditioning with oxidative stress may provide cardioprotection similar to ischaemic preconditioning, against ischaemia-reperfusion injury and this cardioprotective effect may be mediated through activation of PKC.     

Dehydrotrametenolic acid selectively inhibits the growth of H-ras transformed rat2 cells and induces apoptosis through caspase-3 pathway

The screening of natural products that preferentially inhibit growth of H-ras transformed rat2 cells vs. rat2 cells was performed to identify H-ras specific growth inhibitor. A lanostane-type triterpene acid, dehydrotrametenolic acid (3beta-hydroxylanosta-7,9(11),24-trien-21-oic acid), was isolated from the sclerotium of Poria cocos (Polyporaceae). Dehydrotrametenolic acid selectively inhibited the growth of H-ras transformed cells with a GI(50) value of 40 microM. FACS analysis indicated that the compound exerted its anti-proliferation effects through cell cycle arrest at G2/M phase and accumulation of sub-G1 population. Dehydrotrametenolic acid-induced apoptosis was further confirmed with chromosomal DNA fragmentation, caspase-3 activation, and degradation of PARP and Lamin A/C degradation. The compound also regulated the expression of H-ras, Akt and Erk, which are the downstream proteins of H-ras signaling pathways. The results suggest that dehydrotrametenolic acid can be a potential anticancer agent against H-ras transformed tumor.     

A method to quantitatively detect H-ras point mutation based on electrochemiluminescence

Conventional methods for point mutation detection are usually multi-stage, laborious, and need to use radioactive isotopes or other hazardous materials, and the assay results are often semi-quantitative. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electrochemiluminescence (ECL) assay was coupled with restriction endonuclease digestion directly from PCR products. Only the wild-type amplicon containing the endonuclease's recognition site can be cut off, and thus cannot be detected by ECL assay. Using the PCR-ECL method, 30 bladder cancer samples were analyzed for possible point mutation at codon 12 of H-ras oncogene. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than three orders of magnitude. The point mutation was found in 14 (46.7%) out of 30 bladder cancer samples. The experiment results demonstrate that the PCR-ECL method is a feasible quantitative approach for point mutation detection due to its safety, high sensitivity, and simplicity.     

高灵敏度电化学发光PCR方法检测基因点突变研究

近年来发展了一种用于定量检测基因点突变的电化学发光PCR方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对待测基因进行PCR扩增;随后,采用特定的限制性内切酶对扩增产物进行酶切,由于野生型样品和突变型样品间存在酶切位点的变化,其中只有一种基因型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到样品池中;进行电化学发光检测,通过所得信号的有无可以判断其基因型。我们分别将该法用于Presenilin-1基因和H-ras癌基因的点突变检测,结果均可明显区分突变型样品和野生型样品。该法具有灵敏、快速、简便、安全等优点,是一种实用的基因点突变检测方法。     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras^lys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-αp85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.