Analysis of growth dynamics and the relationship to the fluctuations in alkaline phosphatase in two strains of cells of HeLa S3 derivation

Summary Studies have been carried out to determine an association between glucocorticoid-induced changes in the pattern of growth and the fluctuations of alkaline phosphatase in two HeLa strains. The results showed that growth arrest in steroid-treated cells did not have the characteristics of density-induced growth inhibition. Alkaline phosphatase increased with increased cell density, the increase being greater than control in steroid-treated cells of the “inducible” strain (HeLa S3G, HeLa₆₅) and less than control in the “suppressible” strain (HeLa S3K, HeLa₇₁). Increased serum concentration in the growth medium (0 to 20%) caused an increase in alkaline phosphatase in S3G strain and a decrease in the S3K strain. This investigation was supported by the Veterans Administration and by USPHS Research Grant CA-08315 from the National Cancer Institute.     

Induction of human choriogonadotropin in HeLa-cell cultures by aliphatic monocarboxylates and inhibitors of deoxyribonucleic acid synthesis

The ectopic production of the glycopeptide hormone human placental choriogonadotropin by HeLa₆₅ cells was measured by radioimmunoassay with antiserum against the β-subunit of choriogonadotropin and with the ¹²⁵I-labelled β-subunit as a tracer antigen. Choriogonadotropin synthesis was markedly (500-fold) stimulated by sodium butyrate. Kinetic studies and the use of an inhibitor of protein synthesis, cycloheximide, indicated that protein synthesis was required for this induction. Investigation of the efficiency of 22 aliphatic short-chain fatty acids and derivatives in causing increased choriogonadotropin synthesis by HeLa cells showed stringent structural requirements. Induction of choriogonadotropin synthesis in HeLa cells was not restricted to butyrate. Other aliphatic acids (propionate, isobutyrate, valerate and hexanoate) were also capable of inducing choriogonadotropin synthesis at 10–50% of the efficiency of butyrate. Hydroxy derivatives of monocarboxylate inducers, related mono- and di-carboxylic acids, alcohols, amines, ketones, esters and sulphoxide were ineffective in increasing choriogonadotropin production by HeLa cells. A saturated C₄ straight-chain acid without substituent hydroxyl groups but with a methyl group at one end and a carboxyl moiety at the other appeared to be most efficient in activating choriogonadotropin production. A second clonal line of HeLa cells, HeLa₇₁, showed a higher constitutive synthesis of choriogonadotropin than HeLa₆₅ cells, which was also markedly increased by butyrate. Butyrate and other aliphatic monocarboxylate inducers of choriogonadotropin synthesis inhibited HeLa-cell growth and DNA synthesis. This inhibition of DNA replication may be related to the mechanism of choriogonadotropin synthesis, since two well-characterized anti-neoplastic inhibitors of DNA synthesis, hydroxyurea and 1-β-d-arabinofuranosylcytosine, also stimulated a 300-fold increase in choriogonadotropin synthesis in HeLa cells and were synergistic with butyrate in promoting choriogonadotropin synthesis. Thus activation in tumour cells of genes normally expressed by foetal tissue and the consequent ectopic synthesis of polypeptide hormones may require neither cell division nor DNA synthesis.     

Effect of ZnO and TiO2 nanoparticles preilluminated with UVA and UVB light on Escherichia coli and Bacillus subtilis

We evaluated the effects of zinc oxide (ZnO) and titanium dioxide (TiO₂) nanoparticles (NPs) preilluminated with ultraviolet light on Escherichia coli and Bacillus subtilis. The experiments were conducted using three different types of light: visible, Ultraviolet A (UVA, 315–400 nm), and Ultraviolet B (UVB, 280–315 nm). The bacteria were exposed to NPs, either as liquid suspensions for growth inhibition assays or on agar plates for colony forming unit (CFU) assays. We found that the ZnO NPs were more toxic when preilluminated with UVA or UVB light than with visible light in both growth inhibition and CFU assays. TiO₂ NPs were not toxic to the bacteria under UVA or UVB preillumination conditions. The photo-dissolution of ZnO NPs increased with UV preillumination, which could explain the observed toxicity of ZnO NPs. We detected oxidative stress elicited by photoactive nanoparticles by measuring superoxide dismutase activity. The results of this study show that the toxicity of photoactive nanoparticles can be increased by UV preillumination by dissolution of toxic ions, which suggests the potential for preillumination-dependent toxicity of nanoparticles on soil environments in low light or darkness.     

Tailoring the Field Distribution of ZnO by Polyaniline for SPR-Based Fiber Optic Detection of Hardness of the Drinking Water

Due to the industrial development, drinking water is getting polluted by disposing several waste products of the industries. Hardness is one of the prominent impurities in drinking water which is mainly due to the presence of carbonate and bicarbonate ions (CO₃ ^2? and HCO₃ ^?) in it. Here, we present the synthesis of the zinc oxide (ZnO) and polyaniline (PANI) nanocomposite for the detection and estimation of hardness of the drinking water. The chemical formula of such a nanocomposite is defined in terms of the fraction of polyaniline nanoparticles reinforced in ZnO matrix and is derived as ZnO_(1???x)PANI _x (0?≤?x?≤?0.9); x is the composition ratio. Silver and ZnO_(1???x)PANI _x layers are coated over the unclad core of the optical fiber so as to create the four layer system as that of Kretschmann configuration SPR structure. The working principle involves the change in dielectric constant of (ZnO_(1???x)PANI _x ) by CO₃ ^2? or HCO₃ ^? ions in aqueous atmosphere. Due to the strong interaction of the sensing surface to the CO₃ ^2? and HCO₃ ^? ions, a red shift in the SPR spectrum is observed in the concentration range 0–200 μg/l. The sensitivity of the sensor depends on the composition ratio of the nanocomposite and has been found to be maximum for the composition ratio lying in the range 0.45–0.60. This has been further confirmed in terms of the enhancement of the electric field density and found to be in agreement with the experimental value. The sensitivity of the sensor with optimum value of the composition ratio is 0.094 and 0.065 nm/(μg/l) for CO₃ ^2? and HCO₃ ^?, respectively. The sensor is highly selective to CO₃ ^2? and HCO₃ ^?. The sensor has advantages of online monitoring and remote sensing of water quality because the probe is fabricated over an optical fiber.     

Nanostructural zinc oxide hollow spheres: A facile synthesis and catalytic properties

The development of reproducible procedures for the synthesis and organization of nanostructured metal oxides is important in order to exploit the unique properties of these materials for practical applications. The present work describes the transformation of Zn(NH₃)₄]²⁺ into hollow structured ZnO materials through solvothermal decomposition. An increase in ammonia concentration in the reaction medium, significantly changes the morphology of ZnO from spheres made of nanoparticles (20-30 nm) to hollow spheres composed of nanorods (200-350 nm) or to free microrods as evidenced from scanning and transmission electron micrographs (SEM/TEM). The powder X-ray diffraction (XRD) pattern of ZnO confirms formation of the wurtzite structure. Raman and Energy-dispersive spectroscopic (EDS) studies indicate the presence of oxygen deficiency in ZnO. The investigation on the catalytic behavior of ZnO in the synthesis of (4-methoxyphenyl)(phenyl) methanone (MPPM) by Friedel-Crafts acylation of anisole with benzoyl chloride has also been carried out. The results reveal that the prepared ZnO could produce ∼98% of yield compared to 41% produced by commercial ZnO.     

Suberoyl bishydroxamic acid-induced apoptosis in HeLa cells via ROS-independent, GSH-dependent manner

Suberoyl bishydroxamic acid (SBHA) is a HDAC inhibitor that can regulate many biological functions including apoptosis and proliferation in various cancer cells. Here, we evaluated the effect of SBHA on the growth of HeLa cervical cancer cells in relation to apoptosis, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC₅₀ of approximately 15 μM at 72 h. SBHA also induced apoptosis in HeLa cells, as evidenced by sub-G1 cells, annexin V-FITC staining cells, activations of caspase 3 and 8, and the loss of mitochondrial membrane potential (ΔΨ_m). In addition, all of the tested caspase inhibitors rescued some cells from SBHA-induced HeLa cell death. SBHA increased ROS levels including O ₂ ^?? and induced GSH depletion in HeLa cells. Generally, caspase inhibitors did not affect ROS levels in SBHA-treated HeLa cells, but they significantly prevented GSH depletion in these cells. Furthermore, while the well-known antioxidants, N-acetyl cysteine and vitamin C, did not affect cell death, ROS level or GSH depletion in SBHA-treated HeLa cells, l-buthionine sulfoximine, a GSH synthesis inhibitor, enhanced cell death and GSH depletion in these cells. In conclusion, SBHA inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis, and the inhibition is independent of ROS level changes, but dependent on GSH level changes.     

Antiproliferative and Apoptosis Inducing Effects of Non-Polar Fractions from Lawsonia inermis L. in Cervical (HeLa) Cancer Cells

Two non-polar fractions viz. hexane (Hex-LI) and chloroform fraction (CHCl₃-LI) of Lawsonia inermis were studied for their antiproliferative potential in various cancer cell lines viz. HeLa, MCF-7, A549 and C6 glioma cells. Both the fractions showed more than 60 % of growth inhibition in all the tested cell lines at highest tested concentration. In clonogenic assay, different concentrations of Hex-LI and CHCl₃-LI decreased the number and size of colonies as compared to control in HeLa cells. The apoptotic effects as nuclear condensation, fragmentation were visualized with Hoechst-33342 staining of HeLa cells using confocal microscope. Both fractions induced apoptotic cell death in human cervical carcinoma (HeLa) cells as evident from flow cytometric analysis carried out using Annexin V-FITC and propidium iodide dyes. CHCl₃-LI treated cells significantly induced apoptosis (25.43 %) in comparison to control. Results from Neutral Comet assay demonstrated that both fractions induced double stranded breaks (DSB’s) in HeLa cells. Our data indicated that Hex-LI and CHCl₃-LI treated cells showed significant increase of 32.2 and 18.56 % reactive oxygen species (ROS) levels in DCFH-DA assay respectively. Further, experimental studies to decipher exact pathway via which these fractions induce cell death are in progress.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

Cytotoxic effect of Green synthesized silver nanoparticles using Melia azedarach against in vitro HeLa cell lines and lymphoma mice model

This communication explains the biosynthesis of stable silver nanoparticles (AgNPs) from Melia azedarach and its cytotoxicity against in vitro HeLa cells and in vivo Dalton's ascites lymphoma (DAL) mice model. The AgNPs synthesis was determined by UV–visible spectrum and it was further characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and X-ray diffraction (XRD) analysis. Zeta potential analysis revealed stable AgNPs at ?24.9 mV. UV visible spectrum indicated an absorption peak at 436 nm which reflects its specific Surface Plasmon Resonance (SPR). Biosynthesized AgNPs were predominantly cubical and spherical with an average particle size of 78 nm approximately as observed through SEM and DLS analysis, respectively. Cytotoxicity of biosynthesized AgNPs against in vitro Human epithelial carcinoma cell line (HeLa) showed a dose–response activity. Lethal dose (LD₅₀) value was found to be 300 μg/mL of AgNPs against HeLa cell line. Cytotoxicity against normal continuous cell line human breast lactating, donor 100 (HBL 100) was found only in increased concentration of both AgNPs and 5-FU. In addition, in vivo DAL mice model showed significant increase in life span, induction of apoptosis was evidenced by acridine orange and ethidium bromide (AO and EB) staining.     

Design and synthesis of the novel oleanolic acid-cinnamic acid ester derivatives and glycyrrhetinic acid-cinnamic acid ester derivatives with cytotoxic properties

Oleanolic acid (OA) and glycyrrhetinic acid (GA) are natural products with anticancer effects. Cinnamic acid (CA) and its derivatives also exhibited certain anticancer activity. In order to improve the anticancer activity of OA and GA, we designed and synthesized a series of novel OA-CA ester derivatives and GA-CA ester derivatives by using molecular hybridization approach. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess their in vitro cytotoxicity on three cell lines (HeLa (cervical cancer), MCF-7 (breast cancer) and L-O2 (a normal hepatic cell)). Among the evaluated compounds, 3o presented the strongest selective cytotoxicity on HeLa cells (IC₅₀ = 1.35 μM) and showed no inhibitory activity against MCF-7 cells (IC₅₀ > 100 μM) and L-O2 cells (IC₅₀ > 100 μM), and 3e presented the strongest selective inhibition of the MCF-7 cells (IC₅₀ = 1.79 μM). What’s more, compound 2d also showed very strong selective inhibitory activity against HeLa cells (IC₅₀ = 1.55 μM). The further research using Hoechst 33342, AO/EB dual-staining, flow cytometric analysis and DCFH-DA fluorescent dye staining assay presented that 2d and 3o could induce HeLa cells apoptosis and autophagy.     

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin ‘reaction centre’

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O₂. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e₆ (ZnCe₆) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn₂^II,II centre, and that ZnCe₆ binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe₆ initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the Mn^II EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of Mn^III species. The formation of the Mn^III is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.     

Cytotoxic diterpenoids as potential anticancer agents from the twigs of Casearia kurzii

A search for bioactive natural products as anticancer lead compounds has led to the isolation of five new clerodane diterpenoids (1–5) from the twigs of Casearia kurzii. Their structures were elucidated by extensive analysis of their NMR, IR, and HRESIMS data, and the absolute configurations were determined by experimental and calculated electronic circular dichroism (ECD) data analysis. The isolates were biologically evaluated and showed cytotoxic activities toward human lung cancer cells (A549), human cervical cancer cells (HeLa), and human hepatocellular carcinoma cells (HepG2). The most active compound (5) with an IC₅₀ value of 5.3 μM against HeLa cells, was found to induce apoptosis and arrest the HeLa cell cycle at G0/G1 stage to exert cytotoxic effects.     

The Aqueous Extract of Ficus religiosa Induces Cell Cycle Arrest in Human Cervical Cancer Cell Lines SiHa (HPV-16 Positive) and Apoptosis in HeLa (HPV-18 Positive)

Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FR_aq) bark in human cervical cancer cell lines, SiHa and HeLa. FR_aq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G₁/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FR_aq induced apoptosis through an increase in intracellular Ca²⁺ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FR_aq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FR_aq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.     

Plasmon Responses in the Sodium Tungsten Bronzes

The sodium tungsten bronzes (Na _x WO₃) are vividly coloured metallic materials where the optical behaviour can be attributed to the bulk plasma frequency occurring in the visible part of the spectrum. A combination of density functional theory (DFT) calculations and experimental electron energy-loss spectroscopy (EELS) was used to assess their bulk and surface plasmon responses. It was observed that Na _x WO₃ can sustain strong localised surface plasmon resonances (LSPR) with an energy that can be tuned by changing the Na content. They have a stronger plasmonic response when compared to Au and do not suffer from the atmospheric corrosion of Ag, giving them good potential for plasmonic applications.     

Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells

Cervical cancer causes many deaths in females worldwide, including in Indonesia. Several studies have reported that soursop (Annona muricata L.) leaves can be used to treat cervical cancer. This study aims to determine the use of endophytic fungi of A. muricata leaves extract as an ingredient that inhibits cervical cancer. The isolated endophytic fungi from various soursop leave accessions were grown in culture media, then extracted using ethyl acetate. The extract was then tested against anti-yeast, cervical cancer cells, and on normal cells as control using the MTT method. Five isolated fungi were selected based on the greatest inhibition in one concentration, and the inhibitory concentration 50 (IC₅₀) value was determined. The soursop leaves endophytic fungi extracts showed cytotoxicity against cervical cancer cells by inhibiting the multiplication of HeLa cancer cells in vitro. The Sir-SM2 endophytic fungi crude ethyl acetate extract showed high cytotoxicity to cervical cancer cells (HeLa cells) but less harmful to the normal Chang cells; therefore can be a natural anticancer. Identification based on morphology shows that the isolated Sir-SM2 endophytic fungi belong to the Penicillium genus, and molecular identification based on Internal Transcribed Spacer shows high similarities with Penicillium crustosum.     

G-quadruplex inducer/stabilizer pyridostatin targets SUB1 to promote cytotoxicity of a transplatinum complex

Pyridostatin (PDS) is a well-known G-quadruplex (G4) inducer and stabilizer, yet its target genes have remained unclear. Herein, applying MS proteomics strategy, we revealed PDS significantly downregulated 22 proteins but upregulated 16 proteins in HeLa cancer cells, of which the genes both contain a number of G4 potential sequences, implying that PDS regulation on gene expression is far more complicated than inducing/stabilizing G4 structures. The PDS-downregulated proteins consequently upregulated 6 proteins to activate cyclin and cell cycle regulation, suggesting that PDS itself is not a potential anticancer agent, at least toward HeLa cancer cells. Importantly, SUB1, which encodes human positive cofactor and DNA lesion sensor PC4, was downregulated by 4.76-fold. Further studies demonstrated that the downregulation of PC4 dramatically promoted the cytotoxicity of trans-[PtCl₂(NH₃)(thiazole)] (trans-PtTz) toward HeLa cells to a similar level of cisplatin, contributable to retarding the repair of 1,3-trans-PtTz crosslinked DNA lesion mediated by PC4. These findings not only provide new insights into better understanding on the biological functions of PDS but also implicate a strategy for the rational design of novel multi-targeting platinum anticancer drugs via conjugation of PDS as a ligand to the coordination scaffold of transplatin for battling drug resistance to cisplatin.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.