Human Cervical Cells Research Articles

Unusual Enhancement of Doxorubicin Activity on Co-Delivery with Polyhedral Oligomeric Silsesquioxane (POSS).

Polyhedral oligomeric silsesquioxane (POSS), bearing eight 3-chloroammoniumpropyl substituents, was studied as a potential nanocarrier in co-delivery systems with doxorubicin (DOX). The toxicity of doxorubicin and POSS:DOX complexes at four different molar ratios (1:1; 1:2, 1:4, 1:8) towards microvascular endothelial cells (HMEC-1), breast cancer cells (MCF-7), and human cervical cancer endothelial cells (HeLa) was determined. The rate of penetration of the components into the cells, their cellular localization and the hydrodynamic diameter of the complexes was also determined. A cytotoxicity profile of POSS:DOX complexes indicated that the POSS:DOX system at the molar ratio of 1:8 was more effective than free DOX. Confocal images showed that DOX co-delivery with POSS allowed for more effective penetration of doxorubicin through the cell membrane. Taking all the results into account, it can be claimed that the polyhedral oligomeric silsesquioxane (T8-POSS) is a promising, complex nanocarrier for doxorubicin delivery.

Scaffolds based on chitosan/pectin thermosensitive hydrogels containing gold nanoparticles

Thermosensitive hydrogels based on chitosan/pectin (CS/Pec) and CS/Pec/gold nanoparticles (CS/Pec/AuNPs) were successfully prepared with different AuNP levels. Using a tilting method, gelation temperature was demonstrated to decrease when the amount of AuNPs increased and pectin concentrations decreased. The presence of AuNPs in the CS/Pec composite was evaluated via WAXS and UV–vis techniques, while SEM analysis assessed the average size of pores (350–600μm). All samples were extremely cytocompatible with many cell types, such as normal kidney epithelial cells (VERO cells), epithelial colorectal adenocarcinoma cells (HT-29 cells), HPV-16 positive human cervical tumour cells (SiHa cells), kidney epithelial cells (LLCMK2 cells) and murine macrophage cells (J774A1 cells). Cell viability assays using the MTT method upon mouse preosteoblastic cells (MC3T3-E1 cells) showed that CS/Pec and CS/Pec/AuNPs composites had the potential to foster proliferation and growth of bone cells, making them possible stimulators for reconstruction of bone tissues.

Crucial role of estrogen for the mammalian female in regulating semen coagulation and liquefaction in vivo.

Semen liquefaction changes semen from a gel-like to watery consistency and is required for sperm to gain mobility and swim to the fertilization site in the Fallopian tubes. Kallikrein-related peptidases 3 (KLK3) and other kallikrein-related peptidases from male prostate glands are responsible for semen liquefaction by cleaving gel-forming proteins (semenogelin and collagen). In a physiological context, the liquefaction process occurs within the female reproductive tract. How seminal proteins interact with the female reproductive environment is still largely unexplored. We previously reported that conditional genetic ablation of Esr1 (estrogen receptor α) in the epithelial cells of the female reproductive tract (Wnt7aCre/+;Esr1f/f) causes female infertility, partly due to a drastic reduction in the number of motile sperm entering the oviduct. In this study, we found that post-ejaculated semen from fertile wild-type males was solidified and the sperm were entrapped in Wnt7aCre/+;Esr1f/f uteri, compared to the watery semen (liquefied) found in Esr1f/f controls. In addition, semenogelin and collagen were not degraded in Wnt7aCre/+;Esr1f/f uteri. Amongst multiple gene families aberrantly expressed in the absence of epithelial ESR1, we have identified that a lack of Klks in the uterus is a potential cause for the liquefaction defect. Pharmacological inhibition of KLKs in the uterus replicated the phenotype observed in Wnt7aCre/+;Esr1f/f uteri, suggesting that loss of uterine and seminal KLK function causes this liquefaction defect. In human cervical cell culture, expression of several KLKs and their inhibitors (SPINKs) was regulated by estrogen in an ESR1-dependent manner. Our study demonstrates that estrogen/ESR1 signaling in the female reproductive tract plays an indispensable role in normal semen liquefaction, providing fundamental evidence that exposure of post-ejaculated semen to the suboptimal microenvironment in the female reproductive tract leads to faulty liquefaction and subsequently causes a fertility defect.

Heat-Induced Editing of HPV Genes to Clear Mucocutaneous Warts?

Hyperthermia increases expression of the antiviral cellular factors APOBEC3A and APOBEC3G and induces G-to-A or C-to-T mutations in human papilloma virus cervical cell lines and genital warts. This unexpected effect of heat treatment correlated with regression of genital warts in a subset of patients, including at distant sites, suggesting that this effect may be mediated in part by antiviral as well as immunological mechanisms.

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.

Assessment of anti-cancerous potential of 6-gingerol (Tongling White Ginger) and its synergy with drugs on human cervical adenocarcinoma cells

The anti-cancerous activity of 6-gingerol extracted from Tongling White Ginger was investigated. 6-Gingerol inhibited the growth of HeLa cells with IC50 (96.32 μM) and IC80 (133.01 μM) and led to morphological changes, induced the cell cycle arrest in G0/G1-phase and ultimately resulted into apoptosis. Among cell cycle-related genes and proteins, the expression of cyclin (A, D1, E1) reduced, while of CDK-1, p21 and p27 showed slight decrease, except cyclin B1 and E1 (protein). Western blotting reported the induction of apoptosis with an increased Bax/Bcl-2 ratio, release of cytochrome c, cleavage of caspase-3, -8, -9 and PRPP in treated cells. 6-Gingerol activated AMPK, but inhibited PI3K/AKT phosphorylation with reduced P70S6K expression and also suppressed the mTOR phosphorylation. 6-Gingerol with 5-FU and Ptx resulted in 83.2% and 52% inhibition respectively, this synergy have stimulated apoptosis proteins more efficiently as compared to 6-Gingerol alone (10.75%) under in vitro conditions.

Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis.

The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL­1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment.

Association of Casp3 microRNA Target Site (1049216) SNP With the Risk and Progress of Cervical Squamous Cell Carcinoma.

In the present study, we investigated the relationship between the single-nucleotide polymorphism (SNP) of caspase-3 rs1049216 (C > T), a miRNA target site, and the risk and progression of cervical cancer. Using polymerase chain reaction-restriction fragment length polymorphism, we evaluated the genotype and distribution of caspase-3 rs1049216 in 515 patients with cervical squamous cell cancer and 415 controls. In additional experiments, we transfected luciferase reporter plasmids carrying T or C allele and/or miRNA mimics into the human cervical cell lines (HeLa and C-33A) to analyze its roles in the regulation of caspase-3 expression. By immunohistochemistry, the protein level of caspase-3 expression was examined in tumor tissues from 515 patients with cervical squamous cell cancer. We found that the TT genotype of caspase-3 rs1049216 conferred a significantly decreased risk of cervical cancer (adjusted odds ratio, 0.35; 95% confidence interval, 0.154-0.581) and may be associated with the progression of this cancer. Although the expression of caspase-3 in the TT genotype was higher than that in CC/CT genotype in peripheral blood mononuclear cells and tumor tissues. Additional luciferase analysis showed that the rs1049216 variant T allele was associated with significantly higher luciferase activity, compared with the C allele in the transfected cells, and when cotransfected with miRNAs, miRNA-181a could downregulate the luciferase activity in the cells that transfected the construct containing C allele, compared with T allele, which had not happened in the presence of other miRNAs selected. These data indicate that through upregulating the expression of caspase-3, the TT genotype of caspase-3 rs1049216 can be associated with not only the risk of cervical cancer but also the progression of this cancer.

Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells

It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation-induced alteration in mitochondrial function influences tumor cell viability, various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin (Tempol) with TPP+ (named “Mito-”) were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)10-Tempol (M10T) and its derivatives, Mito-(CH2)5-Tempol (M5T), Mito-(CH2)10-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). M10T, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation-induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. M10T treatment inhibited X-ray-induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TPP+ is partly responsible for the observed radiosensitization.

Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3.

Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines.

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

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.

EMMPRIN Inhibits bFGF-Induced IL-6 Secretion in an Osteoblastic Cell Line, MC3T3-E1.

Background: Electrolytically-generated acid functional water (FW) is obtained by electrolyzing low concentrations of saline. Although it has been widely used in clinical practice with various purposes, the underlying mechanisms of action involved have not been fully elucidated so far. We used the human cervical cancer-derived fibroblastic cell line (HeLa), to examine the cytokine secretion profile following FW treatment in the present study.Results: FW stimulation significantly induced the secretion of basic fibroblast growth factor (bFGF) and extracellular matrix metalloproteinase inducer (EMMPRIN). The effect of both factors on osteoblast-like MC3T3-E1 cells was further examined by stimulating the cells with the conditioned medium of FW-stimulated HeLa cells. However, the conditioned medium failed to induce IL-6 secretion. The MC3T3-E1 cells were further stimulated with recombinant bFGF or EMMPRIN or a combination of both factors. Intriguingly, bFGF-stimulated IL-6 induction was totally inhibited by EMMPRIN. Pretreatment with the specific inhibitor of nuclear factor-kappa B (NF-κB) drastically inhibited IL-6 secretion indicating that bFGF-induced IL-6 expression was dependent on NF-κB activation. The phosphorylation status of NF-κB p65 subunit was further examined. The results indicated that EMMPRIN inhibited bFGF-induced NF-κB p65 phosphorylation.Conclusions: These findings suggest that bFGF can induce IL-6 secretion in MC3T3-E1 cells through NF-κB activation. As EMMPRIN inhibited bFGF-induced IL-6 secretion by reducing the p65 subunit phosphorylation, it might be concluded that bFGF and EMMPRIN crosstalk in their respective signaling pathways.

Expression of OCT-4 in cervical cancer patients with different radiosensitivity and effect of OCT-4 knockdown on radiosensitivity of cervical cancer cells

Objective To study the expression of OCT-4 in tumor tissues of cervical cancer patients with different radiosensitivity, and to explore the effect of OCT-4 knockdown on radiosensitivity of human cervical squamous carcinoma Siha cells and its mechanism. Methods Real-time PCR and Western-blot were used to determine the mRNA and protein expression of OCT-4, respectively, in tumor tissues of cervical cancer patients with different radiosensitivity. The Siha cells were transfected with OCT-4-SiRNA to construct the OCT-4 knockdown cell line (OCT-4 SiRNA group). The control group and NC transfection group were also set up. The Siha cells in each group were exposed to different doses of X-ray. The MTT assay and colony formation assay were used to evaluate cell proliferation and colony formation ability in each group, respectively. Flow cytometry was used to assess apoptosis and cell cycle in each group. Western blot was used to measure the expression of cleaved caspase-3 and p-Akt in each group. Results With the decrease in radiosensitivity of patients with cervical cancer, the mRNA and protein expression of OCT-4 in tumor tissues were significantly increased (P<0.05). Compared with the control group and the NC group, the OCT-4 SiRNA group had a significantly increased cell proliferation inhibition rate, significantly weaker colony formation ability, significantly enhanced apoptosis and cell cycle inhibition, significantly higher expression of cleaved caspase-3, and significantly lower expression of p-Akt after exposure to different doses of X-ray (all P<0.05). Conclusions OCT-4 expression is negatively correlated with the radiosensitivity of patients with cervical cancer. OCT-4 knockdown enhances the radiosensitivity of human cervical squamous carcinoma Siha cells by promoting the expression of cleaved caspase-3 and inhibiting the expression of p-Akt. Key words: OCT-4 gene; Cervical neoplasms/radiotherapy; Siha cells line; OCT-4 gene silence; Radiosensitivity

Effects of microRNA-34a overexpression on radiosensitivity of four human cervical cancer cell lines

Objective To investigate the effects of miRNA-34a overexpression on the radiosensitivity of four human cervical cancer cell lines. Methods Cervical cancer lines, C33A, HeLa, SiHa, and CaSki, were transfected with pGenesil-1-miRNA-34a plasmid using the liposome 2000 kit. Cervical cancer lines overexpressing miRNA-34a (miRNA-34a/C33A, miRNA-34a/HeLa, miRNA-34a/SiHa, and miRNA-34a/CaSki) were constructed by G418 selection. These cells were exposed to different doses of 60Coγ-ray. The expression of miRNA-34a was determined using TaqMan real-time PCR and Western blot. The cell growth inhibition rate and colony formation ability were measured by MTT assay and colony formation assay, respectively. Apoptosis and expression of apoptosis-related proteins were evaluated using flow cytometry and Western blot, respectively. Results The expression of miRNA-34a was significantly higher in positive cervical cancer cell lines than in the negative control groups (P<0.05). The cervical cancer cell lines overexpressing miRNA-34a had significantly elevated growth inhibition rates and significantly weaker colony formation ability than the negative control groups (both P<0.05). The results of flow cytometry showed that cervical cancer cell lines overexpressing miRNA-34a had significantly higher apoptosis rates than the negative control groups (P<0.05). The results of Western blot indicated that cervical cancer lines overexpressing miRNA-34a had increased expression of cleaved caspase 9 and cleaved PARP proteins after exposure to 4 Gy+ radiation. Conclusions Cervical cancer lines overexpressing miRNA-34a are successfully constructed in this study. Overexpression of miRNA-34a enhances the radiosensitivity of cervical cancer cells. The mechanism is related to the activation of apoptosis pathway. Key words: miRNA-34a; Cervical neoplasm cell line; Radiosensitivity; Cell growth; Apoptosis

ANP32A Regulates Histone 3 Acetylation and Promotes Leukemogenesis in AML

Leukemia initiation and progression not only depend on genetic alterations but also on abnormal epigenetic changes that cause disorder of gene expression. Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) binds directly to histone tails, preferentially to unmodified histone H3 tails (Schneider et al., 2004; Seo et al., 2002). Although ANP32A inhibits histone acetyltransferase activity in vitro (Seo et al., 2002), loss of ANP32A leads to decreased histone 3 acetylation (acetyl-H3) levels on interferon-stimulated genes in human cervical carcinoma HeLa S3 cells (Kadota et al., 2011). Aberrant ANP32A expression has been observed in many solid tumors, which involves in diverse processes depending on the cell context (Brody et al., 2004; Hoffarth et al., 2008), yet mechanisms are still unknown. Furthermore, there has as yet been no specific investigation into the role of ANP32A in acute myeloid leukemia (AML).We found that ANP32A expression was upregulated at both mRNA and protein levels in patients with AML. In AML primary cells from patients, ANP32A KD decreased cell proliferation and colony formation ability. In addition, ANP32A KD induced apoptosis and cell cycle arrest at G1 phase accompanied with the downregulated protein expression of survival or cell cycle progress genes BCL2, CDK4, CCND1 and the upregulated protein expression of pro-apoptosis genes BAK, BAD, cleaved caspase 3. These findings were also verified in various subtypes of AML cell lines and Mll-Af9 pre-leukemic cells. Thus, ANP32A is essential for cell growth via the promotion of cell-cycle progression and inhibition of apoptosis in AML.Given that ANP32A associates with histone acetylation modification, we analyzed the effect of ANP32A depletion on acetyl-H3 and histone 4 acetylation (acetyl-H4). Western blot analysis showed that ANP32A KD caused global decrease of acetyl-H3, but no effect on the global level of acetyl-H4. To investigate the mechanism of ANP32A in AML, ChIP-seq and gene expression microarray experiments were performed to determine acetyl-H3 enrichment and transcriptome profiles, respectively, in ANP32A KD and control K562 cells. We found a positive correlation between changes in gene expression and changes in acetyl-H3 (r=0.16, p<10-4), especially for differential expression genes (DEGs; r=0.40, p<10-4). Moreover, we also found genes with decreased acetyl-H3 enrichment displayed lower expression levels compared with genes with increased acetyl-H3 enrichment (p<10-3). Thus, ANP32A alters acetyl-H3 modification in association with changes in gene expression.Gene set enrichment analysis (GSEA) revealed that ANP32A KD significantly decreased the acetyl-H3 enrichment levels at genes of lipid metabolism signatures (e.g. lipid metabolic process, cholesterol homeostasis). Transcriptome profile analysis using GSEA showed that gene expression levels of lipid metabolic process signature were also reduced after ANP32A KD. We further identified 4 genes (APOC1, CNKSR3, H19, PCSK9) which had significantly lower levels of mRNA expression (p<10-2, FDR<10-2, FC>1.5) and decreased levels of acetyl-H3 enrichment (p<10-2, FDR<10-2, FC>1.5) in ANP32A KD cells. Apolipoprotein CI(APOC1) was the most significantly downregulated gene among these 4 genes, with decreased acetyl-H3 on its promoter. To explore whether lipid metabolism as a downstream effector of ANP32A contributes to ANP32A function in AML, re-introduction of APOC1 was performed in ANP32A KD THP1 or K562 cells. Re-introduction of APOC1 significantly rescued the inhibition of cell growth in ANP32A KD AML cells, also upregulated protein expression of BCL2, CDK4 and CCND1, showing that ANP32A functions in AML partially depending on APOC1. Importantly, APOC1 depletion decreased cell proliferation of AML cells in vitro, consistent with the point of view that complex lipid alterations contribute to endoplasmic reticulum stress-induced apoptosis in cancer cells (Beloribi-Djefaflia et al., 2016; Iurlaro et al., 2016).Overall, our data suggest ANP32A as a novel regulator of leukemogenesis in AML. ANP32A may involve in the regulation of genome-wide histone 3 acetylation that alters the expression of multiple genes, including lipid metabolism related gene APOC1. Thus, ANP32A is a promising drug target for AML therapy. DisclosuresNo relevant conflicts of interest to declare.

Methodology for monitoring gold nanoparticles and dissolved gold species in culture medium and cells used for nanotoxicity tests by liquid chromatography hyphenated to inductively coupled plasma-mass spectrometry

An analytical methodology based on coupling reversed-phase liquid chromatography (HPLC) to an inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the characterization and identification of gold nanoparticles (AuNPs) and gold dissolved species (Au3+) in culture medium (Dulbecco's Modified Eagle Medium, DMEM) and HeLa cells (a human cervical adenocarcinoma cell line) used in nanotoxicity tests. The influence of the culture medium was also studied and the method applied for nanotoxicity tests. It was also observed that AuNPs can undergo an oxidation process in the supernatants and only a small amount of AuNPs and dissolved Au3+ was associated with cells. To evaluate the biological impact of AuNPs, a classical viability assay onto HeLa cells was performed using cellular media DMEM in the presence of increasing dosage of 10nm AuNPs. The results showed that 10nm AuNPs exhibit a slight toxic effect.

Effect of SP600125 on the mitotic spindle in HeLa Cells, leading to mitotic arrest, endoreduplication and apoptosis.

BackgroundThe JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking mitosis progression and inducing cell death. Despite, all this study, the mechanism by which SP600125 inhibits mitosis-related effects in human cervical cells (HeLa cells) remains unclear. In this study, we investigated the effects of SP600125 on the cell viability, cell cycle, and on the spindle assembly during mitosis in HeLa cells.MethodsTo explore this approach, we used a viability test, an immunofluorescence microscopy to detect Histone phosphorylation and mitotic spindle aberrations. Apoptosis was characterised using Western Blotting.ResultsTreatment of HeLa cells with varying concentrations of SP600125 induces significant G2/M cell cycle arrest with elevated phosphorylation of histone H3 within 48 h, and endoreduplication after 48 h. SP600125 also induces significant abnormal mitotic spindle. High concentrations of SP600125 (20 μM) induce disturbing microtubule assembly in vitro. Additionally, SP600125- induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activation in the late phase (at 72 h).ConclusionOur results confirmed that SP600125 induce mitosis arrest in G2/M, endoreduplication, mitotic spindle aberrations and apoptosis.

Eulophia macrobulbon – an orchid with significant anti-inflammatory and antioxidant effect and anticancerogenic potential exerted by its root extract

BackgroundThe Orchidaceae family is one of the largest families of flowering plants. Orchids are widely used for the traditional herbal medicine, acting as aphrodisiac, antisepic, antimicrobial, anti-cancer agent, etc. PurposeThis study was designed to elucidate the anti-inflammatory, antioxidant and cytotoxic potential of a 50% ethanolic extract of Eulophia macrobulbon roots (EME) in vitro, an orchid growing in Southern Asia. Furthermore, the main active compounds were isolated, and the bioactivity of the single constituents was determined. MethodsThe anti-inflammatory activity of EME and its compounds was evaluated by the secretion of pro- and anti-inflammatory cytokines and by the expression of inducible nitric oxide synthase (iNOS) in a lipopolysaccharide (LPS)-stimulated macrophage model, as determined by an enzyme linked immunosorbent assay (ELISA) and Western blot. Antioxidant activity was assessed using a DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) photometric assay. Cytotoxic effects were determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. ResultsEME and its compounds significantly reduced the production of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), the expression of iNOS and subsequently increased the production of the anti-inflammatory cytokine interleukin 10 (IL-10) in LPS-stimulated macrophages. Additionally it could be demonstrated that EME is rich in radical scavengers. Furthermore, EME and its components showed notable cytotoxic effects on the human cervical adenocarcinoma cell line HeLa, the human colorectal adenocarcinoma cell line CaCo-2 and the human breast adenocarcinoma cell line MCF-7. The most active constituents were identified as 4-methoxy-9,10-dihydro-2,7-phenanthrenediol (8), 4-methoxy-2,7-phenanthrenediol (9), 1,5-dimethoxy-2,7-phenanthrenediol (10), 1,5,7-trimethoxy-2,6-phenanthrenediol (11), 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrenediol (15). ConclusionBased on this data, EME provides various beneficial anti-inflammatory, antioxidant and cytotoxic attributes and may be used as herbal remedy in the pharmaceutical or food industries.

Effects of sirolimus and starved culture on autophagy of human A431 cells

Objective To evaluate effects of sirolimus (a classic autophagy inducer) and starved culture on autophagy of a human cutaneous squamous cell carcinoma cell line A431. Methods Cultured A431 cells and HeLa (a human cervical carcinoma cell line) cells were both classified into 5 groups to be treated with DMEM alone (control group) , 0.1% dimethyl sulfoxide alone (DMSO group) , 20 nmol/L sirolimus (20-nmol/L sirolimus group) , 80 nmol/L sirolimus (80-nmol/L sirolimus group) , and Earle′s balanced salt solution (EBSS group) respectively. After 4-hour treatment, Western blot analysis was performed to measure the expressions of autophagy-related markers microtubule-associated protein 1 light chain 3A/3B (LC3A/B) and recombinant gamma-aminobutyric acid receptor associated protein (GABARAP) , and acridine orange staining to determine autophagy levels in these cells. Results As Western blot analysis showed, the ratio of LC3A/B -Ⅱ to LC3A/B -Ⅰ in A431 cells was similar between the control group and DMSO group (P > 0.05) , but significantly higher in the 20-nmol/L sirolimus group, 80-nmol/L sirolimus group and EBSS group than in the control group (all P < 0.05). Western blot results from HeLa cells were similar to those from A431 cells. Bivariate correlation analysis revealed that the protein expression of GABARAP was positively correlated with that of LC3A/B -Ⅰ in both HeLa cells (r = 0.869, 95% CI: 0.807-0.999, P= 0.051) and A431 cells (r= 0.837, 95% CI: -0.173-0.989, P= 0.037) , but negatively correlated with that of LC3A/B-Ⅱ in both HeLa cells (r=-0.742, 95% CI: -0.982-0.406, P = 0.042) and A431 cells (r=- 0.684, 95% CI: -0.977-0.500, P = 0.047). Acridine orange staining showed that the percentages of autophagosome-positive A431 cells and HeLa cells were significantly increased in both the 80-nmol/L sirolimus group (23.750% ± 0.260% and 33.307% ± 0.715% respectively) and EBSS group (32.450% ± 0.488% and 66.097% ± 1.141% respectively) compared with the control group (15.987% ± 0.242% and 14.117% ± 0.295%, respectively, all P < 0.05) . Conclusion The classic autophagy inducer sirolimus and starved culture can upregulate the autophagy level of A431 cells, and GABARAP may be highly correlated with LC3A/B. Key words: Autophagy; Cell line, tumor; HeLa cells; Sirolimus; Hunger; A431 cells; GABARAP