Human Cervical Cell Line HeLa Research Articles

Validation of mitotic harvesting method with human cervical carcinoma HeLa cells expressing fluorescent ubiquitination-based cell cycle indicators for radiation research.

The cell cycle is a series of events in the process of one cell giving rise to two daughter cells. The mitotic harvesting method, established by Terasima and Tolmach in the 1960s, causes minimal physiological stress on the cells and achieves a high degree of cell cycle synchrony by collecting only mitotic cells from a cultured cell system. The purpose of the present study is to validate the versatility of the mitotic harvesting method using human cervical cell line HeLa cells expressing Fluorescent Ubiquitination-based Cell Cycle Indicators (FUCCI) and to estimate the cell cycle-dependent changes in radiosensitivity in HeLa-FUCCI cells. The image analysis showed that cell cycle synchrony was maintained for at least 24 hours after mitotic cell collection. Also, the clonogenic assay demonstrated changes in radiosensitivity that were cell cycle dependent. These results indicate that the mitotic harvesting method using FUCCI-expressing cells has high versatility in the field of radiation cell biology.

Combination of temsirolimus with cisplatin as anti-cancer agents through inducing apoptosis and altering cell cycle distribution in Hela cell line

Combination treatment is the most effective therapy strategy on cancer by dramatically reducing drug toxicity and enhancing efficacy. In this study, we assessed whether temsirolimus, an mTOR inhibitor enhanced anti-cancer effects of cisplatin on human cervical Hela cell line or not. The combination of temsirolimus and cisplatin, to a large extent, reduced proliferation rate of cervical Hela cells in comparision with single chemotherapeutics cisplatin in vitro and resulted in synergistic effects (CI<1). Flow cytometry results indicated that temsirolimus markedly increased cisplatin-induced apoptosis. Thus, the combination chemotherapy using cisplatin and temsirolimus could be a novel strategy for synergistic effects against human cervical cancer.

Herpes simplex virus type 2 inhibits TNF-α-induced NF-κB activation through viral protein ICP22-mediated interaction with p65.

Herpes simplex virus type 2 (HSV-2) is a prevalent human pathogen and the main cause of genital herpes. After initial infection, HSV-2 can establish lifelong latency within dorsal root ganglia by evading the innate immunity of the host. NF-κB has a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. It is known that inhibition of NF-κB activation by a virus could facilitate it to establish infection in the host. In the current study, we found that HSV-2 inhibited TNF-α-induced activation of NF-κB-responsive promoter in a dose-dependent manner, while UV-inactivated HSV-2 did not have such capability. We further identified the immediate early protein ICP22 of HSV-2 as a vital viral element in inhibiting the activation of NF-κB-responsive promoter. The role of ICP22 was confirmed in human cervical cell line HeLa and primary cervical fibroblasts in the context of HSV-2 infection, showing that ICP22 deficient HSV-2 largely lost the capability in suppressing NF-κB activation. HSV-2 ICP22 was further shown to suppress the activity of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase α (IKK α)-, IKK β-, IKK γ-, or p65-induced activation of NF-κB-responsive promoter. Mechanistically, HSV-2 ICP22 inhibited the phosphorylation and nuclear translocation of p65 by directly interacting with p65, resulting in the blockade of NF-κB activation. Furthermore, ICP22 from several alpha-herpesviruses could also inhibit NF-κB activation, suggesting the significance of ICP22 in herpesvirus immune evasion. Findings in this study highlight the importance of ICP22 in inhibiting NF-κB activation, revealing a novel mechanism by which HSV-2 evades the host antiviral responses.

Differentiation inducing factor-1 suppressed the epithelial-mesenchymal transition via degradation of Yes-associated protein

Differentiation-inducing factors (DIFs), produced by Dictyostelium discoideum, show anti-tumor activity via several different signaling molecules, including glycogen synthase kinase-3 (GSK-3) and signal transducer and activator of transcription 3 (STAT3). However, the exact mechanism of action of DIFs is still poorly understood. Therefore, to better understand the action of DIF, we performed DNA microarray analysis and found that the activity of the Hippo signaling pathway may be modified. Therefore, we analyzed mRNA expressions of Hippo signaling pathway target genes. Although the mRNA expression of the target genes including CTGF and Cyr61were elevated by DIF-1, surprisingly the protein expression themselves were reduced. In an attempt to elucidate the mechanism, we found that DIF promoted proteolysis of Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signaling pathway, in the human cervical cell line HeLa and the human colon cancer cell line HCT-116. YAP has also been reported to promote epithelial-to-mesenchymal transition (EMT). Indeed, cell migration, cell invasion and expressions of EMT-related proteins (fibronectin, vimentin and N-cadherin) were reduced by DIF-1. These results suggest that DIF-1 suppresses EMT via degradation of YAP in human cancer cell lines.

Synthesis and Anti Cervical Cancer Activity of Novel 5H-Thiochromeno [4,3-d]pyrimidines

A series of novel 5H-Thiochromeno[4,3-d]pyrimidine derivatives were synthesized, purified and characterized by different spectroscopy techniques such as 1H NMR, 13C NMR, Mass and Elemental Analysis. The new compounds were evaluated for their anti-cervical cancer activity on Human Cervical Cell Line HeLa. They were found to be potent anti-cervical cancer agents with GI50 values less than 10 μg/mL with respect to positive control drug Adriamycin.

Fabrication of nanoparticles from a synthesized peptide amphiphile as a versatile therapeutic cargo for high antiproliferative activity in tumor cells.

Nanoparticles with encapsulated small molecules have attained vital importance in anticancer research. Peptide-based nanoparticles show their versatility in drug delivery due to their excellent biocompatibility and nontoxic nature. We demonstrate here the design and fabrication of peptide-based nanoparticles as dual-therapeutic cargo for the controlled release of hydrophilic 5-Fluorouracil (5Fu) and hydrophobic camptothecin (CPT), simultaneously. The covalent conjugation of 5Fu with the peptide, through a stimuli-responsive linker, provided better control over the release of 5Fu and dramatically reduced the possibility of leaching of the small molecule. As anticipated, the peptide-5Fu nanoparticles were efficient to encapsulate a second chemotherapeutic molecule CPT in its hydrophobic region. The stimuli-responsive release of 5Fu was carefully monitored by HPLC, NMR, and UV-visible spectroscopy. On the other hand, the release of the hydrophobic drug CPT from the nanoparticles was determined to be in a diffusion-controlled fashion. Assessment of performance in human cervical HeLa cell lines demonstrated the peptide-drug nanoparticles to be highly nontoxic. Whereas, the simultaneous release of the two antitumor agents, in a controlled manner, resulting in rapid antiproliferation of the tumor cells.

Inhibition of Key Enzymes Linked to Obesity and Cytotoxic Activities of Whole Plant Extracts of Vernonia mesplilfolia Less

The whole plant of Vernonia mespilifolia is widely used as a traditional remedy for obesity in South Africa. The aim of this study was to investigate the anti-obesity and cytotoxic effects of Vernonia mespilifolia extracts in vitro. The α-amylase, α-glucosidase, and lipase inhibitory activities of aqueous and ethanol extracts of Vernonia mespilifolia were investigated, while the cytotoxic effects of these extracts were analyzed using Hoechst 33342 and propidium iodide (PI) dual staining on a human cervical HeLa cell line. The results showed that the LC50 (the concentration of a material will kill 50% of test organisms) values of aqueous and ethanol extracts of Vernonia mespilifolia were >200 and 149 µg/mL, respectively, to HeLa cells. Additionally, the ethanol extract exhibited the strongest inhibitory effect on the pancreatic lipase (Half-maximal inhibitory concentration (IC50) = 331.16 µg/mL) and on α-amylase (IC50 = 781.72 µg/mL), while the aqueous extract has the strongest α-glucosidase (IC50 = 450.88 µg/mL). Our results suggest that Vernonia mespilifolia’s acclaimed anti-obesity effects could be ascribed to its ability to inhibit both carbohydrate and fat digesting enzymes.

Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice

Human cervical cancer is the fourth most common carcinoma in women worldwide. However, the emergence of drug resistance calls for continuously developing new anticancer drugs and combination chemotherapy regimens. The present study aimed to investigate the anti-cervical cancer effects of metformin, a first-line therapeutic drug for type 2 diabetes mellitus, and nelfinavir, an HIV protease inhibitor, when used alone or in combination. We found that both metformin and nelfinavir, when used alone, were moderately effective in inhibiting proliferation, inducing apoptosis and suppressing migration and invasion of human cervical cell lines HeLa, SiHa and CaSki. When used in combination, these two drugs acted synergistically to inhibit the growth of human cervical cancer cells in vitro and cervical cancer cell xenograft in vivo in nude mice, and suppress cervical cancer cell migration and invasion. The protein expression of phosphoinositide 3-kinase catalytic subunit PI3K(p110α), which can promote tumor growth, was remarkably downregulated, while the tumor suppressor proteins p53 and p21 were substantially upregulated following the combinational treatment in vitro and in vivo. These results suggest that clinical use of metformin and nelfinavir in combination is expected to have synergistic antitumor efficacy and significant potential for the treatment of human cervical cancer.

The restraining effect of baicalein and U0126 on human cervical cancer cell line HeLa.

To explore the restraining effect of baicalein and the mitogen-activation protein kinase kinase inhibitor, U0126, on human cervical cell line HeLa proliferation, apoptosis and migration. HeLa cells were treated by different concentrations of baicalein or U0126. A Cell Counting Kit (CCK)‑8 assay was applied to examine cell viability. Flow cytometry was used to determine cell cycle and apoptosis. A wound healing assay was performed to detect cell migration. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was adopted to test cell apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis was used to detect apoptosis gene and protein expression. CCK‑8 assay demonstrated that baicalein and U0126 suppressed HeLa cell viability by dose dependence. TUNEL, Annexin V‑fluorescein isothiocyanate/propidium iodide, and ratio of Bcl‑2‑associated X protein and B cell lymphoma 2 indicated that baicalein and U0126 induced HeLa cell apoptosis. Flow cytometry revealed that baicalein blocked the cell cycle of HeLa in G0/G1 phase. A wound healing assay demonstrated that baicalein significantly inhibited HeLa cell migration compared with control. Baicalein and U0126 markedly downregulated extracellular signal‑regulated kinase 1/2, matrix metalloproteinase (MMP) 2 and MMP9 levels both in mRNA and protein. In the present study, the authors demonstrated that baicalein and U0126 may be used in cervical cancer treatment by inhibiting cell migration and inducing cell apoptosis.

Chemical Composition, Cytotoxic and Antibacterial Activities of the Essential Oil from the Tunisian Ononis angustissima L. (Fabaceae).

The chemical composition, cytotoxic and antibacterial activities of the hydrodistilled essential oil of the aerial parts of Ononis angustissima from south Tunisia has been evaluated. The oil yield was 0.04% (w/w). The chemical composition, determined by GC and GC-MS is reported for the first time. Forty-five components, accounting for 93.7% of the total oil have been identified. The oil was characterized by a high proportion of oxygenated sesquiterpenes (33.2%), followed by sesquiterpene hydrocarbons (6.3%) and apocarotenoids (10.3%). The main components of the oil were α-eudesmol (22.4%), 2-tridecanone (9.3%) and acetophenone (7.4%). The essential oil was tested for its possible cytotoxic activity towards the human cervical cell line HeLa using the MTT assay and the antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and the clinical strain Acinetobacter sp. This oil exerted a cytotoxic activity with an IC50 of 0.53 ± 0.02 mg/mL and a significant antibacterial effect against P. aeruginosa and E. faecalis.

Possible roles of LC3-independent autophagy in elimination of Shigella in human colon cells (INC4P.343)

Abstract Autophagy is essential for the defense against bacterial invasion. The intestinal tract is quite specialized in order to co-exist with extracellular bacteria, and thus, this system efficiently exploits autophagy to regulate the number of bacteria and to control the immune response to preventing chronic inflammation. Bacterial elimination is mediated by microtubule-associated protein 1A/1B-light chain 3 (LC3), which is critical for the activation of the autophagic canonical pathway in mammals. Although both human colon HT-29 cell line and the human cervical HeLa cell line display normal LC3 expression, HeLa cells are very susceptible to Shigella infection, whereas HT-29 cells efficiently restrict the number of intracellular Shigella. Our results suggest that colon cells may utilize LC3-independent noncanonical autophagy to regulate bacterial growth and survival.

Phenethyl isothiocyanate upregulates death receptors 4 and 5 and inhibits proliferation in human cancer stem-like cells.

BackgroundThe cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.MethodsCancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers’ analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.ResultsPEITC attenuated proliferation of CD44high/+/CD24low/–, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.ConclusionsThe anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Differentiation-Inducing Factor-1 Suppresses the Expression of c-Myc in the Human Cancer Cell Lines

Differentiation-inducing factor-1 (DIF-1), a morphogen for Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by suppressing the Wnt/β-catenin signaling pathway. In this study, we examined the effect of DIF-1 on c-Myc, a target gene product of the Wnt/β-catenin signaling pathway, mainly using HCT-116 colon cancer cells. DIF-1 strongly reduced the amount of c-Myc protein in time- and concentration-dependent manners and reduced c-Myc mRNA expression by inhibiting promoter activity through the TCF binding sites. The effect of DIF-1 on c-Myc was also confirmed using the human cervical cell line HeLa. Pretreatment with the proteasome inhibitor MG132 or glycogen synthase kinase-3β (GSK-3β) inhibitors (LiCl and SB216763) attenuated the effect of DIF-1, suggesting that DIF-1 induced c-Myc protein degradation through GSK-3β activation. Furthermore, we examined whether c-Myc was involved in the anti-proliferative effect of DIF-1 using c-Myc–overexpressing cells and found that c-Myc was associated with the anti-proliferative effect of this compound. These results suggest that DIF-1 inhibits c-Myc expression by inhibiting promoter activity and inducing protein degradation via GSK-3β activation, resulting in the inhibition of cell proliferation. Since c-Myc seems to be profoundly involved in accelerated proliferation of various malignant tumors, DIF-1 may have a potential to develop into a novel anti-cancer agent.

INDUCTION OF APOPTOSIS IN CERVIX NEOPLASMS HeLa CELLS BY A RAPESEED PEPTIDE HYDROLYSATE FRACTION

One rapeseed peptide fraction (RSP2) was prepared from rapeseed meal by enzyme digestion and separation on Sephadex gel-filtration column. The anticancer effects of RSP2 on human cervical HeLa cell lines were evaluated. Exposure of RSP2 caused a significant decrease in viability of HeLa cells in a dose-dependent manner and typical morphological changes and apoptosis bleb phenomenon in RSP2-treated HeLa cells were observed. DNA fragmentation assay and single-cell gel electrophoresis showed that RSP2 could induce apoptosis in HeLa cells at all concentrations tested in a dose-dependent manner. Together with our recently published data that RSP2 could decrease the percentage of HeLa cells in the G0/G1phase from 55% to 43%, and increase the ratio of apoptotic cells from 2.9 to 40%, results of this study highlighted the candidacy of RSP2 as a novel source of biologically active peptides with an inhibiting effect on human cancer cells. PRACTICAL APPLICATIONS Apoptosis is an important biological mechanism to maintain the integrity of multicellular organisms and the failure of apoptosis could lead to the development of human malignancies. Rapeseed is the world's second leading source of protein meal and could potentially be used as a food ingredient instead of being wasted or being used for animal feed. In this study, we identified one rapeseed peptide fraction prepared from rapeseed meal as a source of bioactive peptides with anticarcinogenic properties on human cancer cell through the induction of apoptosis. In addition, our previous study showed rapeseed peptide hydrolysate may be useful as a human food addition with antioxidant properties. Therefore, an underutilized by-product such as defatted rapeseed meal could be utilized as novel therapeutic agents to fight against cancer and develop new functional foods.

Microarray Analysis Identifies Differentially Expressed Genes Induced by Human Papillomavirus Type 18 E6 Silencing RNA

The oncoprotein E6 of high-risk human papillomavirus (HPV) types promotes cell proliferation and contributes to carcinogenesis of HPV-positive cervical cancer cells. In this study, we used small interfering RNA (siRNA) technology to silence the E6 gene in HPV-18-transformed human cervical cell line HeLa and determined the effects of E6 gene knockdown on the cell by using microarray-based gene expression profiling coupled with gene functional classification with bioinformatics methods. Silencing RNA prepared by siRNA expression cassettes against HPV-18 E6 gene could significantly inhibit E6 gene expression and induce HeLa cells to apoptosis. The microarray analysis identified 359 differentially expressed genes containing 307 up-regulated and 52 down-regulated genes. We analyzed the gene functions and cellular pathways in detail, including cell cycle-related genes, CCNG1 and p21; apoptosis-related genes, CASP4, CASP6, IGFBP3, and DFFA; ubiquitin proteolysis pathway-related genes, UBE3A and UBE2C; keratinocyte differentiation-related genes, KRT4, KRT6E, and KRT18; and antioncogenes, RECK and VEL. In addition, it can be concluded that cellular apoptosis induced by HPV-18 E6 siRNA mainly depends on the P53 and ubiquitin proteolysis pathway to regulate gene expression, consequently inhibiting cell proliferation and promoting cell apoptosis. Meanwhile, activation of antioncogene and upper regulation of immunization-related genes signified the degression of the malignant extent of tumor cells after E6 inhibition. Our approach, which combines the use of siRNA-mediated gene silencing, microarray screening, and functional classification of differential genes, can be used in functional genomics study to elucidate the role of E6 oncogene in the carcinogenesis of HPV-18 and provide some possible targets for clinical treatment and drug development of cervical cancer.