HeLa Cell DNA Research Articles

Cervical cancer gene therapy by gene loaded PEG-PLA nanomedicine.

Advances in the treatment of cervical cancer over the last decade have predominantly involved the development of genes directed at molecular targets. Gene therapy is recognized to be a novel method for the treatment of cervical cancer. Genes can be administered into target cells via nanocarriers. This study aimed to develop systemically administrable nano-vectors. Floate (Fa) containing gene loaded nanoparticles (NPs) could target HeLa human cervical cancer cells through combination with receptors on the cells to increase the nuclear uptake of genetic materials. Fa was linked onto Poly (ethylene glycol)-b-poly (D, L-lactide) (PEG-PLA) to form Fa-PEG-PLA, and the resulting material was used to load plasmids of enhanced green fluorescence protein (pEGFP) to obtain gene loaded nanoparticles (Fa-NPs/DNA). Physical-chemical characteristics, in vitro release and cytotoxicity of Fa-NPs/DNA were evaluated. The in vitro transfection efficiency of Fa-NPs/ DNA was evaluated in HeLa cells and human umbilical vein endothelial cells (HUVEC). PEG-PLA without Fa was used to load pEGFP from NPs/DNA as a control. Fa-NPs/DNA has a particle size of 183 nm and a gene loading quantity of 92%. After 72h of transfection, Fa-NPs/DNA displayed over 20% higher transfection efficiency than NPs/DNA and 40% higher than naked DNA in HeLa cells. However, in HUVECs, no significant difference appeared between Fa-NPs/DNA and NPs/DNA. Fa-PEG-PLA NPs could function as excellent materials for gene loading. This nano-approach could be used as tumor cell targeted medicine for the treatment of cervical cancer.

HOGG1-Cys326 variant cells are hypersensitive to DNA repair inhibition by nitric oxide

The repair of 8-oxo-7,8-dihydroguanine in the DNA of mammalian cells is initiated by 8-oxoguanine DNA glycosylase (OGG1). A frequent polymorphism in the human OGG1 gene, rs1052133, causes the substitution of serine by cysteine at amino acid 326 of the protein and has been associated with an altered risk for various types of cancer in some populations. The OGG1-Cys326 protein appears to have normal enzymatic activity, but greater sensitivity to oxidation than the serine variant. Here, we describe a comparison of the cellular repair by the two OGG1 variants under stress conditions characteristic of inflammation, namely in cells pretreated with nitric oxide (NO) or pre-exposed to hyperthermia. The results show that NO at concentrations causing negligible DNA damage and little cytotoxicity strongly reduces the repair rates of oxidized purines in the DNA of HeLa cells overexpressing the OGG1-Cys326 variant. The reduction in repair was much less pronounced in isogenic cells overexpressing the OGG1-Ser326 variant. Similar results were observed in EBV-transformed lymphocytes from donors homozygous for the two OGG1 variant alleles. In contrast, hyperthermia-induced stress caused a repair retardation that was independent of the OGG1 polymorphism. The repair inhibition by NO in the variant cells gave rise to increased genetic instability, measured as increased micronuclei formation after oxidant exposure. The results could explain a higher risk of malignant transformation in inflamed tissues of carriers of this variant allele.

Human papillomavirus in esophageal squamous cell carcinoma

Context: We studied the role of Human papillomavirus (HPV) in esophageal squamous cell carcinoma from North East India. Methods: Fifteen archival paraffin embedded esophageal squamous cell carcinoma tissues were retrieved. Thin sections were made to extract DNA from the tissues. HeLa cell lines which harbor HPV DNA were used as a positive control. Nested polymerase chain reaction (PCR) was performed to analyze the presence of HPV DNA. Amplified products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining. Positive product was sequenced for confirmation of the result. Results: HPV DNA was not able to detect in any esophageal squamous cell carcinoma tissue. Positive control HeLa cells DNA was found to be positive for HPV DNA by nested PCR. Conclusions: Our study shows the negative association of HPV in esophageal squamous cell carcinoma. However, further studies are needed with a large number of sample size to rule out the role of HPV in this region.

Daunomycin, an antitumor DNA intercalator, influences histone–DNA interactions

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25–250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

Synthesis, crystal structure, interaction with DNA, and cytotoxicity in vitro of a new mixed ligand-nickel complex: [Ni(DBMA)(en)(H2O)3]·3H2O

A mixed ligand-nickel complex of formula [Ni(DBMA)(en)(H2O)3]·3H2O, where DBMA = 2,2-dibenzyl malonic acid, en = ethylenediamine, has been synthesized and characterized by elemental analysis, IR, and single-crystal X-ray diffraction. The unit cell parameters for the complex are a = 16.0214(16) Å, b = 10.0801(10) Å, c = 15.4024(15) Å, β = 108.488(2)°, V = 2359.074(40) Å3, Z = 4, space group P2(1)/c. The crystal system of the complex is monoclinic. Thermal stability of the water cluster by thermogravimetric analysis has been studied. Gel electrophoresis assay demonstrated the ability of the complex to cleave pBR322 plasmid DNA. The binding of the complex with HeLa cell DNA (HC-DNA) has been investigated by fluorescence spectra. Value of IC50 calculated for the complex shows that the complex exhibits good cytotoxicity against HeLa and KB cell lines.

Effect of propofol on mitochondrial DNA in HeLa cells

Effect of propofol on mitochondrial DNA in HeLa cells

A bioreducible polypeptide for efficient gene transfection both in vitro and in vivo

A disulfide bond containing polypeptide, PolyK6-R8, was designed and prepared by oxidative polymerization between terminal cysteinyl thiol groups of CHK6HC and CR8C. The molar ratio between CHK6HC and CR8C within obtained PolyK6-R8 was 9:1, and PolyK6-R8 could combine DNA compactly when weight ratio reached 5. Through in vitro investigation, it was found that PolyK6-R8 was an efficient gene vector with low cytotoxicity for delivering DNA in both COS-7 and HeLa cells. Cellular uptake of DNA mediated by PolyK6-R8/DNA complexes was promoted after incubation for 4 h. Moreover, by examining the histological sections of hindlimb ischemia rats through immunohistochemistry, PolyK6-R8/VEGF complexes were proved to be effective in both VEGF protein expression and succeeding vessel formation. The results indicated that polypeptide-based PolyK6-R8 is a promising gene vector for the limb ischemia treatment.

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30 bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.

Quercetin induces cytochrome‐c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug‐DNA interaction

Small aromatic compounds like flavonoids can intercalate with DNA molecules bringing about conformational changes leading to reduced replication and transcription. Here, we have examined one dietary flavonoid, quercetin (found in many fruit and vegetables), for possible anti-cancer effects, on HeLa cells originally derived from a case of human cervical cancer. By circular dichroism spectroscopy we tested whether quercetin effectively interacted with DNA to bring about conformational changes that would strongly inhibit proliferation and migration of the HeLa cells. Cytotoxic effects of quercetin on cancer/normal cells, if any, were determined by MTT assay and such depolarization of mitochondrial membrane potential, as a consequence of quercetin treatment, and accumulation of reactive oxygen species (ROS) also were studied, by FACS analysis and expression profiles of different anti- and pro-apoptotic genes and their products were determined. Quercetin intercalated with calf thymus cell DNA and HeLa cell DNA and inhibition of anti-apoptotic AKT and Bcl-2 expression were observed. Levels of mitochondrial cytochrome-c were elevated and depolarization of mitochondrial membrane potential occurred with increase of ROS; upregulation of expression of p53 and caspase-3 activity were also noted. These alterations in signalling proteins and externalization of phosphotidyl serine residues were involved with initiation of apoptosis. Reduced AKT expression suggested reduction in cell proliferation and metastasis potential, with arrest of the cell cycle at G2/M. Quercetin would have potential for use in cervical cancer chemotherapy.

Cytotoxic Effect and Induction of Apoptosis in Human Cervical Cancer Cells by Antrodia camphorata

Antrodia camphorata is a Chinese herb indigenous to Taiwan. Previous reports demonstrated that it could induce apoptosis in some cancer cells. The purpose of this study was to investigate the apoptotic effect of the crude extract of A. camphorata in cervical cancer cells. Two human cervical cancer cell lines, HeLa and C-33A, were treated with extract of A. camphorata (10-1000 μg/mL). We found that A. camphorata extract was cytotoxic to both cervical cancer cells in a dose- and time-dependent manner as examined by MTT assay. Treatment with A. camphorata extract at 400 μg/mL induced a 2.3- and 4.4-fold increase in oligonucleosome formation from the cleaved chromosomal DNA in HeLa and C-33A cells, respectively. A. camphorata extract also activated caspase-3, -8, and -9 activities and increased the cytosolic level of cytochrome c in both cell lines as the dosage increased. Furthermore, A. camphorata extract increased expressions of Bak, Bad and Bim, while decreasing expressions of Bcl-2 and Bcl-xL of the Bcl-2 family proteins in HeLa and C-33A cells. The expression of IAP proteins, XIAP and survivin, was also decreased in both cervical cancer cells after treatment with A. camphorata. Our in vitro study suggests that A. camphorata is cytotoxic to cervical cancer cells through both extrinsic and intrinsic apoptotic mechanisms. It could be used as a novel phytotherapeutic agent or auxiliary therapy in the treatment of cervical cancer.

Synthesis and chemotherapeutic activities of 5-chloro-1H-indole-2,3-dione-3-thiosemicarbazones

A series of 5-chloro-1H-indole-2,3-dione 3-thiosemicarbazones 3a-g were syn- thesized to investigate the chemotherapeutic activities. The structures of 3a-g were con- firmed by the spectral data (IR, 1H NMR, 13C NMR-APT, 13C NMR-DEPT, HSQC, HMBC) and elemental analysis. Anticancer activities of the compounds were evaluated using cell kinetic parameters on HeLa cells derived from human cervix carcinoma. The preliminary screening results indicated that alkyl substituted compounds 3a, 3b and 3d were more effective in mi- tosis phase when compared to the synthesis phase. 3a-g were tested against some DNA and RNA viruses in CRFK, HeLa, HEL, MDCK and Vero cells. None of the compounds was active against any of the RNA or DNA viruses at 100 μM. All compounds were also evaluated for antimicrobial activity against selected strains. Methyl substituted 3a and allyl substituted 3c were found to be active against S. aureus and C. albicans.

New cytosine derivatives as inhibitors of DNA methylation

DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. Ki values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 μM. These compounds cause also a decrease of 5-methylcytosine (m5C) level in DNA of mammalian HeLa and HEK293 cells.

Two new palladium(ii) complexes: synthesis, characterization and their interaction with HeLa cells

Two complexes [Pd(L(1))(en)](2)·4H(2)O (1), [Pd(L(2))(en)]·2H(2)O (2), (L(1) = pyridine-2,3-dicarboxylic acid, L(2) = pyrazine-2,3-dicarboxylic acid, and en = ethylenediamine) have been synthesized and characterized using IR, (1)H NMR and element analysis. X-ray analyses of complexes 1 and 2 revealed that 1 belongs to the triclinic system with space group P1(2), and 2 belongs to the monoclinic system with space group P2(1)/n. The binding of the complexes with HC-DNA (HeLa cells DNA, which was extracted by ourselves) were measured by fluorescence spectrum and viscometry. Gel electrophoresis assay demonstrates the ability of the complexes to cleave the extracted HC-DNA. The complexes exhibit a higher cytotoxicity against tumor cells as against normal cells in vitro. Further more, apoptotic tests indicate that the complexes have an apoptotic effect on HeLa cells.

Picolinate ruthenium(II)–arene complex with in vitro antiproliferative and antimetastatic properties: Comparison to a series of ruthenium(II)–arene complexes with similar structure

In our previous study, ruthenium(II)-p-cymene complexes of general formula [(η6-p-cymene)Ru(L)Cl2], L: 3-acetylpyridine (1), 2-amino-5-chloropyridine (2); and [(η6-p-cymene)Ru(HL)Cl], HL: 2,3-pyridinedicarboxylic acid (3), 2,4-pyridinedicarboxylic acid (4), revealed low antiproliferative activity, except complex [(η6-p-cymene)RuCl(picolinic acid)]·H2O (5) which exhibited IC50 around 80μM. In this study we further investigated in vitro potential of antimetastatic action of ruthenium complexes on HeLa and two endothelial cell lines. Comparison of structure and activity of five complexes indicated heterogenic mode of activity, with regard to the potential of antimetastatic and antiproliferative effect. Replacement of substituted pyridine ligand with picolinic acid (complex 5) around Ru(II) center contributed to complex cytotoxicity and ruthenium DNA binding affinity. Analysis of ruthenium(II) accumulation in DNA and protein fractions of HeLa cells, using ICP-OES revealed significantly higher content of complex 5 in DNA fraction in comparison to the other tested compounds. It also altered cell cycle progression, affected expression of DNA repair enzymes ERCC1 and MSH2, and showed enhanced activity in combination with 3-aminobenzamide.Regardless of their effect on cell growth, Ru(II) complexes exerted antimetastatic effect on several tumor cell lines in vitro, achieved mostly by the effect on cell adhesion, migration and angiogenesis, while picolinate ruthenium(II)–arene additionally exerted inhibitory effect on extracellular matrix degradation.

Mixed-ligand manganese(II)-phenolate complexes: study of DNA cleavage, cytotoxic activity, and induction of apoptosis

Two manganese complexes, [Mn(L1)2(dibe)2](L1) (1) and [Mn(L2)2(dibe)2] (2) (where L1 = 2,2′-bipyridine (bipy), L2 = 1,10-phenanthroline (phen)), were synthesized by using a carboxylic acid ligand (dibe = 2,2-dibenzylmalonate acid). The two complexes were characterized using IR, elemental analysis, and X-ray crystallography. Fluorescence analysis indicates that the two complexes can bind to HeLa cell DNA (HC-DNA), and gel electrophoresis assay demonstrates the ability of the complexes to cleave the HC-DNA. The two complexes exhibit cytotoxic specificity and significant cancer cell inhibitory rate. Furthermore, apoptotic tests demonstrate that these two complexes have apoptotic effects on HeLa cells.

Novel palladium(II) complexes containing a sulfur ligand: structure and biological activity on HeLa cells

Two novel palladium(II) complexes with a thiosalicylic acid (HSC(6)H(4)CO(2)H) ligand, with the formulas [Pd(TSA)(L)]·mH(2)O (TSAisthiosalicylic acid; in complex 1, Lis1,10-phenanthroline and m=1; in complex 2, Lis2,2'-bipyridine and m=2), have been synthesized and characterized. The coordination geometry of both palladium atoms is square planar; they are four-coordinated and each is coordinated in an N,N,O(-),S(-) mode. There is a sigmoid oxygen chain in complex 1, but an oxygen ring in complex 2. The competitive binding of the complexes to HeLa cell DNA (HL-DNA) has been investigated by fluorescence spectroscopy. The results show that the two complexes have the ability to bind with HL-DNA. Viscosity studies suggest that the complexes bind to DNA by intercalation. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the HL-DNA. The two complexes exhibit cytotoxic specificity and a significant cancer cell inhibitory rate. The apoptosis tests indicated that the complexes have an apoptotic effect. Furthermore, complex 1 exhibits more biological activity than complex 2, which is mainly because the area of the aromatic ring of 1,10-phenanthroline is larger than that of 2,2'-bipyridine.

Synthesis, characterization, and study on HeLa cells activity of a dinuclear complex [Cu4(phen)4(H2O)2]·(pyri)·3H2O

The complex [Cu4(phen)4(H2O)2]·(pyri)·3H2O(where phen = 1,10-phenanthroline and pyri = 3,5-pyridine dicarboxylic acid)has been synthesized and characterized. IR spectra, elemental analysis and X-ray single-crystal diffraction were carried out to determine the composition and crystal structure of the complex. The binding of the complex with HC-DNA (HeLa cells DNA, which was extracted by ourselves) was investigated by fluorescence spectrum. Gel electrophoresis assay demonstrates the ability of the complex to cleave the extracted HC-DNA. Additionally, the complex exhibited a significant cytotoxic specificity and cancer cell inhibitory rate. The apoptotic tests indicate that the complex have an apoptotic effect on HeLa cells.

In vivo toxicity study of N-1-sulfonylcytosine derivatives and their mechanisms of action in cervical carcinoma cell line

New N-1-sulfonylpyrimidines showed potent growth inhibitory activity against human and mouse tumour cells of different origin. 1-(p-toluenesulfonyl)cytosine (TsC) and 1-(p-toluenesulfonyl)cytosine hydrochloride (TsC × HCl) inhibited the growth of human cervical carcinoma cells (HeLa), and had no significant cytotoxic effects on normal human foreskin fibroblasts (BJ). TsC and TsC × HCl interfered with the HeLa cell cycle progression bringing about the accumulation of G1 phase cells and the induction of apoptosis. Antiproliferative effects of TsC and TsC × HCl were additionally confirmed by investigating de novo synthesis of RNA, DNA and proteins in HeLa cells. Monitoring gene expression using DNA Chip Analysis and quantitative PCR showed that TsC × HCl affects the expression of several cell-cycle regulating genes implying that cell cycle arrest and DNA damage-induced apoptosis might account for the observed cellular effects. In vivo experiments revealed low toxicity of TsC × HCl, as demonstrated by unaltered haematological and metabolic blood parameters. In conclusion, potent antitumour efficacy and low toxicity of new compounds in comparison with the common chemotherapy drug 5-FU make them promising anticancer agents. Additional pre-clinical and clinical studies are warranted to illuminate the mode of action of these newly synthesized compounds in vivo, which would lay the groundwork for their further optimization.

Macromolecular profiling of apoptosis via a multiplex biophotonic platform

Traditional fluorescence microscopy is limited to visualizing labeled molecules and does not fully answer questions about molecular composition or cell/tissue organization. As all cells are composed of thousands of individual types of macromolecule, an understanding of the structural organization and specific mutual interactions between macromolecules is important for development in the biomedical sciences and applied medicine. Major vital cellular functions and substructures—such as cytoplasmic organelles—rely upon interactions between proteins, nucleic acids, saccharides, and lipids. Thus, for a comprehensive understanding of how the cell is organized and its function on a molecular level, knowledge of all types of macromolecular distribution, spatial dynamics, and transformations is required. A multiplex biophotonic platform, recently developed by our group, allows for the quantitative characterization and realtime monitoring of local molecular environments in the structural domains of live cells. We combined coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence (TPEF) nonlinear optical imaging with fluorescence recovery after photobleaching (FRAP) and Raman spectroscopy to study the process of apoptosis, a programmed cell death essential for physiological regulation and elimination of genetic disorders.1 While the ability to combine nonlinear imaging has been demonstrated before in the pioneering CARS work of the Sunney Xie group at Harvard University,2 we applied the CARS/TPEF combination for quasi-synchronous multiplex imaging (multiple pictures in one scan) of proteins, lipids, DNA, and RNA during apoptosis.1 Proteins and lipids were distinguished by CARS, while acridine orange-labeled nucleic acids by TPEF responded in green and red spectral channels. CARS/TPEF Figure 1. The distribution of proteins, lipids, DNA, and RNA in HeLa cells during apoptotic development as visualized using multimodal coherent anti-Stokes Raman scattering (CARS) and two-photon excitedfluorescence (TPEF) imaging over 24h. Proteins (red) and lipids (grey) were observed in the CARS mode at the characteristic vibrations of 2928cm 1 and 2890cm 1, respectively. Nucleic acids, stained with acridine orange, were acquired in the red (RNA= green) and green (DNA= blue) fluorescence channels in TPEF mode.

Synthesis and crystal structure of two new dinuclear cobalt(II) complexes interaction with HeLa cells

Two novel dinuclear complexes [Co2(L)2(phen)2·2H2O]n (1), [Co2(L)2(bipy)2]n (2) (where phen=1,10-phenanthroline, bipy=2,2′-bipyridine, and L=biphenyl-2,4′-dicarboxylic acid) have been synthesized and characterized using IR, 1H NMR, element analysis and single-crystal X-ray diffractometry. The binding of the complexes with HC-DNA (HeLa cells DNA, which was extracted by ourselves) was investigated by fluorescence spectrum. The experimental results show that the two complexes have the ability to bind with extracted HC-DNA. Gel electrophoresis assay demonstrates the ability of the complexes to cleave the extracted HC-DNA. The complexes exhibit a higher cytotoxicity against tumor cells as against normal cells in vitro. Further more, the apoptotic tests indicate that the complexes have an apoptotic effect on HeLa cells.