HeLa Proliferation Research Articles

Exploring MiR-484 Regulation by Polyalthia longifolia: A Promising Biomarker and Therapeutic Target in Cervical Cancer through Integrated Bioinformatics and an In Vitro Analysis.

MiR-484, implicated in various carcinomas, holds promise as a prognostic marker, yet its relevance to cervical cancer (CC) remains unclear. Our prior study demonstrated the Polyalthia longifolia downregulation of miR-484, inhibiting HeLa cells. This study investigates miR-484's potential as a biomarker and therapeutic target in CC through integrated bioinformatics and an in vitro analysis. MiR-484 levels were analyzed across cancers, including CC, from The Cancer Genome Atlas. The limma R package identified differentially expressed genes (DEGs) between high- and low-miR-484 CC cohorts. We assessed biological functions, tumor microenvironment (TME), immunotherapy, stemness, hypoxia, RNA methylation, and chemosensitivity differences. Prognostic genes relevant to miR-484 were identified through Cox regression and Kaplan-Meier analyses, and a prognostic model was captured via multivariate Cox regression. Single-cell RNA sequencing determined cell populations related to prognostic genes. qRT-PCR validated key genes, and the miR-484 effect on CC proliferation was assessed via an MTT assay. MiR-484 was upregulated in most tumors, including CC, with DEGs enriched in skin development, PI3K signaling, and immune processes. High miR-484 expression correlated with specific immune cell infiltration, hypoxia, and drug sensitivity. Prognostic genes identified were predominantly epidermal and stratified patients with CC into risk groups, with the low-risk group showing enhanced survival and immunotherapeutic responses. qRT-PCR confirmed FGFR3 upregulation in CC cells, and an miR-484 mimic reversed the P. longifolia inhibitory effect on HeLa proliferation. MiR-484 plays a crucial role in the CC progression and prognosis, suggesting its potential as a biomarker for targeted therapy.

The antitumor effect of mycelia extract of the medicinal macrofungus Inonotus hispidus on HeLa cells via the mitochondrial-mediated pathway.

Inonotus hispidus (I. hispidus), known as shaggy bracket, has been used extensively in China and some East Asian countries as a traditional medicinal macrofungus to treat difficult diseases, such as diabetes, gout, and arthritis. Modern pharmacological research has shown that I. hispidus has an important application value in antitumor treatment. However, the main anti-cervical cancer activity substances from its mycelia and its mechanisms are still not clear. To enrich the germplasm resources of I. hispidus, to reveal the antitumor activity of the extract from the mycelium of I. hispidus against cervical cancer, and to preliminarily analyze its action mechanism. The SH3 strain was isolated from wild fruiting bodies and identified by morphology and molecular biology. The antitumor active component from the mycelium of I. hispidus was isolated and identified with liquid chromatography-tandem mass spectrometry. The cell viability was assessed by MTT assay. The cell cycle distribution, apoptotic cell detection, and mitochondrial membrane potential were detected by flow cytometer. The expression of apoptosis-related proteins was assessed by Western blotting. The inhibition of tumor growth in vivo was assessed by a mouse xenograft model. The SH3 strain was isolated and identified as a new strain of I. hispidus. The antitumor active component containing cyclic peptides from the mycelium of I. hispidus (CCM) was isolated for the first time. In addition, we found that CCM had a strong inhibitory effect on HeLa proliferation in vitro and in vivo. Mechanically, the CCM blocked the cell cycle at the G0/G1 phase, decreased the mitochondrial membrane potential, and eventually promoted apoptosis of HeLa cells through the mitochondria-mediated pathway by upregulating the expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3 and downregulating the expression level of Bcl-2. Our study not only enriches the strain resources of I. hispidus but also confirms that the mycelium of this strain has active components that can inhibit cervical cancer. This is highly significant for the development of active drugs and drug lead molecules for treating cervical cancer.

Extremely low-frequency magnetic fields significantly enhance the cytotoxicity of methotrexate and can reduce migration of cancer cell lines via transiently induced plasma membrane damage

Extra Low-frequency Magnetic Fields (ELF-MFs) significantly enhance cellular uptake of methotrexate by inducing transient plasma membrane pores/damage. This enhanced ‘dose loading’ of methotrexate via the electromagnetically induced membrane pores leads to similar outcomes as the normal control while using significantly smaller therapeutic doses in vitro when compared to non-ELF-MF treated control. Approximately 10% of the typical therapeutic dose yielded similar results when used with ELF-MF. ELF-MFs increase PC12, THP-1 and HeLa proliferation in vitro (120% of the control). Analysis of adherent cells demonstrate significantly less migration towards an induced scratch injury (20 μm in 24 h when compared to a control). Our results suggest an important role for the use of ELF-MFs in the treatment of tumours that opens some new and exciting possibilities including using smaller therapeutic doses of chemotherapeutic agents and disrupting tumour metastasis.

1α,25(OH)2D3(VD3) promotes Raddeanin A-induced anti-proliferative effects on HeLa cell apoptosis and autophagy through negative regulation of HPV18E6-E7/PD-L1/VDR axis

ABSTRACT Raddeanin A (RA) has indicated suppressive effects on various human tumor cells, and insufficient vitamin D was associated with human papillomavirus (HPV) persistence and gynecological tumors. However, combined effects of RA and vitamin D on HPV-positive cells remain elusive. Herein, we aimed to investigate the combined effects of RA and 1ɑ,25(OH)2D3 (VD3) on cellular viability and modulation of HPV18E6/E7, programmed cell death 1 ligand (PD-L1) and vitamin D receptor (VDR) expression in HeLa cells in vitro. HeLa cells were treated with RA alone or VD3 combined with RA. Cell viability was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and apoptosis was detected by flow cytometry. Real-time PCR (qRT-PCR) and Western blot were used to determine the gene/protein expression levels. The autophagosomes were observed by Transmission electron microscopy (TEM). The result showed that cell viability was inhibited by RA, and apoptosis in HeLa cells treated with RA was elevated accordingly. The expression of Bax, Cleaved-caspase-3, Cleaved-caspase-9 and Cleaved-PARP increased, and Bcl-2 decreased. The autophagy was induced by RA, as evidenced by elevated autophagosomes and the increased LC3-II/I ratio and Beclin-1. The expression of HPV18E6/E7, PD-L1 and VDR was reduced by RA. Moreover, RA combined with VD3 had a stronger effect on HeLa cells than RA alone. In conclusion, RA inhibits HeLa proliferation and induces apoptosis and autophagy via suppressing HPV18E6/E7, PD-L1 and VDR, and VD3 showed reinforced effects of RA on HeLa cells. Therefore, combined usage of VD3 with RA might be a potential novel immunotherapy strategy for HPV-related diseases.

Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg2+. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C8 for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.

The Therapeutic Properties of Plants Used Traditionally to Treat Gastrointestinal Disorders on Groote Eylandt, Australia.

The First Australians had well-developed healing systems. Groote Eylandt inhabitants used a variety of plant species to treat diarrhoea and other gastrointestinal illnesses. This study was undertaken to test, identify, and evaluate traditional medicines to treat these conditions against gastrointestinal bacterial, protozoal, and viral pathogens, as well as against cancer cell proliferation. Six plant species (Buchanania obovata Engl., Casuarina equisetifolia L., Eucalyptus tetrodonta F. Muell., Planchonia careya (F. Muell.) R. Knuth, Terminalia carpentariae C. T. White, and Vigna vexillata (L.) A. Rich.) were selected from a survey of a panel of elders from the Warnindhilyagwa tribe and compared with the published literature. Decoctions prepared according to traditional methods were screened for growth inhibitory activity of a panel of diarrhoea-causing bacterial pathogens by disc diffusion and liquid dilution MIC assays. Inhibitory activity against the gastrointestinal protozoal parasite Giardia duodenalis and antiproliferative activity against human colorectal (Caco2) and cervical (HeLa) cancer cell lines were evaluated using MTS-based colorimetric cell proliferation assays. Preliminary antiviral screening was accomplished using an MS2 bacteriophage plaque reduction assay. Toxicity was evaluated using Artemia franciscana nauplii mortality and HDF cell viability bioassays. All traditional medicines tested inhibited bacterial growth, often with MIC values substantially <1000 μg/mL. T. carpentariae was particularly noteworthy, with MIC values of 230–350 μg/mL against Citrobacter freundii, Salmonella newport, Shigella sonnei, Staphylococcus aureus, and Staphylococcus epidermidis. This species also had MICs 450–950 μg/mL against all other bacterial pathogens. B. obovata Engl. and E. tetrodonta were also good inhibitors of bacterial growth, albeit with substantially higher MIC values than determined for T. carpentariae. The T. carpentariae decoction was also the best inhibitor of MS2 phage replication (IC50 = 427 μg/mL) and Caco2 and HeLa proliferation (IC50 values of 885 and 85 μg/mL, respectively). None of the extracts were particularly strong inhibitors of Giardia duodenalis growth. All decoctions were nontoxic in the Artemia nauplii and HDF cell viability bioassays, indicating their suitability for therapeutic use.

Baicalein blocked cervical carcinoma cell proliferation by targeting CCND1 via Wnt/β-catenin signaling pathway

The purpose of this study was to investigate the inhibitory effect of baicalein on the proliferation of cervical carcinoma cells and stimulate cervical carcinoma cells with baicalein. MTT method was used to observe cell proliferation. Flow cytometry was used to observe cell cycle, and gene technology was used to observe the expression of corresponding genes at the level of gene and protein. β-catenin activity was assessed using Western blot and ChIP. Baicalein suppressed cervical carcinoma cell HeLa proliferation by enhancing the activity of caspase-3. Baicalein blocked cell cycle at G0/G1 stage by inhibiting the expression of some genes. At the same time, it can prevent the nuclear translocation of β-catenin and inhibit the activity of Wnt. When the Wnt signaling pathway is increased, the proliferation of HeLa cells is inhibited, and apoptosis is promoted in this way. In conclusion, it indicated that baicalein inhibits cervical carcinoma progression by targeting CCND1 via Wnt/β-catenin signaling pathway.

Deletion of caveolin scaffolding domain alters cancer cell migration

ABSTRACTCaveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT-3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.

Amidated Scolopin‐2 inhibits proliferation and induces apoptosis of Hela cells in vitro and in vivo

This study aimed to investigate the effect of Scolopin-2, a cationic antimicrobial peptide from centipede venoms, and amidated Scolopin-2 on Hela cell viability in vitro and in vivo. The cellular proliferation was investigated with the MTT assay. Confocal laser scanning, flow cytometry, and Western blot analysis were employed to localize Scolopin-2-NH2 in Hela cells and to study the caused cells apoptosis. We subcutaneously injected Hela cells into BALB/c nude mice and studied if Scolopin-2-NH2 suppressed tumor growth in the mice. Scolopin-2-NH2 inhibited Hela proliferation in vitro in a dose-dependent manner with an IC50 of 35 μM. In addition, Scolopin-2-NH2 combined with mitochondria and regulated caspase-related apoptosis pathways in Hela cells. Scolopin-2-NH2 significantly suppressed tumor growth in the tumor-bearing mice without side effects, such as weight loss or abnormal changes in tissues, including liver, spleen, kidney, and lung. These results indicate Scolopin-2-NH2 may be a good therapeutic candidate for the treatment of Hela cervical cancer.

Abstract 3476: Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence

Abstract Telomerase, the enzyme responsible for the length maintenance of telomeres is expressed in about 85% of cancer cells, being a key to tumorigenesis. Telomerase inhibitor drugs have been studied as a promising cancer therapy due to its specificity, with less side effects than conventional approach. However, the effects expected from these therapy require many cell divisions, and the consequences of these long-term therapies are poorly understood. This work evaluated the effects of a long term treatment in cancer cells (HeLa) using BRACO-19, a G-quadruplex-interactive molecule, evaluating the consequences of its use in cell viability and its potential to induce senescence. Telomerase positive HeLa cells were grown in DMEM medium supplemented with 10% fetal bovine serum at pattern conditions. Culture medium was changed every 2 days. For preliminary citotoxicity assay, cells were seeded in 96 wells plate at 3.2x104 cells/cm2, incubated for 24 hours and treated for 24 hours with increasing concentrarions of BRACO-19 and vehicle (0.1% H2O). Cell viability was measured by MTT assay. For the long-term treatment, a subtoxic concentration was chosen. The cells (5.5x102 cells/cm2) were grown under continuous presence of the drug or 0.02% H2O for 11 weeks. During the long-term exposure to BRACO-19, the cells were splitted every 7 - 20 days, depending on its proliferation rate, viable and unviable cells were counted at these points through trypan blue dye exclusion assay. β-Galactosidase (β-gal) activity was used as cell senescence marker to evaluate the senescence after 24 hours treatment and after a long exposure to the telomerase inhibitor. Changes in cell morphology were accessed by phase contrast microscopy. The short-term (24h) treatment with BRACO-19 showed a dose-dependent effect on cell viability. The IC50% for HeLa was 5.25 µM. The subtoxic concentration of 1µM was used for long-term treatment, showing effect on cell proliferation in a way that HeLa cells exposed to the drug showed their proliferative profile decreased when compared to control. Furthemore, no senescent cells were found in control group, even after many passages; on the other hand, 24 hours and long-term exposure to BRACO-19 led to senescence. Long-term treatment was also associated with visual changes on cell morphology. The telomerase inhibitor BRACO-19 showed an expected dose-dependent effect on HeLa cells at short-term exposition, since cytotoxic effect of this compound has been demonstrated in several cell lines, but the impact of the long-term treatment on cell proliferation pattern points BRACO-19 as a potential prototype of a new telomere related drug for cancer therapy. Also, it was demonstrated the senescence inducing action of this drug, perhaps due to erosion of HeLa telomeres. These results show that HeLa is sensitive do BRACO-19 even under subtoxic concentrations. Its effectiveness against other cell lines and tumors is under investigation. Citation Format: Ana Flavia Reis Guimaraes, Diêgo M. de Oliveira. Effect of long term treatment with BRACO 19 on HeLa proliferation and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2017-3476

Inhibition of Caco-2 and HeLa proliferation by Terminalia carpentariae C. T. White and Terminalia grandiflora Benth. extracts: Identification of triterpenoid components

Inhibition of Caco-2 and HeLa proliferation by Terminalia carpentariae C. T. White and Terminalia grandiflora Benth. extracts: Identification of triterpenoid components

Inhibition of Caco-2 and HeLa proliferation by Terminalia sericea Burch. ex DC. (Combretaceae) leaf extracts and identification of volatile components

Terminalia sericea Burch. ex. DC (Combretaceae) is used in traditional South African medicine to treat a wide variety of diseases. Several other Terminalia spp. have been reported to have anti-proliferative and apoptotic activities. Despite this, the anticancer properties of T. sericea remain to be rigorously tested. The current study was undertaken to test a panel of T. sericea leaf extracts for the ability to inhibit the proliferation of Caco-2 colorectal and HeLa cervical carcinoma cells, and to identify volatile compounds using GC-MS headspace analysis. Powdered T. sericea leaves were extracted with solvents of varying polarity and investigated for the ability to inhibit the proliferation of Caco-2 and cervical carcinoma cells using MTS based colorimetric cell proliferation assays. The extracts were tested for toxicity in the Artemia nauplii bioassay and the extract with the most potent anti-proliferative activity was examined using headspace GC-MS analysis to identify volatile nonpolar components. The T. sericea leaf extracts displayed potent anti-proliferative activity against Caco-2 and HeLa carcinoma cells. The methanolic extracts were particularly potent inhibitors of Caco-2 and HeLa proliferation. The aqueous and ethyl acetate extracts (but not the chloroform or hexane extracts) also significantly inhibited carcinoma proliferation, albeit with substantially lower potency. Subsequent analysis of the T. sericea leaf extracts by GC-MS headspace analysis highlighted several interesting volatile compounds. All extracts were shown to be non-toxic in the Artemia nauplii bioassay. The lack of toxicity of these extracts and their anti-proliferative bioactivity against Caco-2 and HeLa carcinoma cells indicates their potential in the treatment of some cancers.

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.

ATM kinase inhibitor KU-55933 contribution in cisplatin mediated HeLa proliferation

Several approaches including chemotherapy and radiation therapies are being at the forefront to treat various types of cancer including cervical cancer. However, the success and failure of genotoxic based therapy is attributed to aberrant ability of carcinoma to patch up genomic breaks. Here, we have used cisplatin as a genotoxic drug model and HeLa as in vitro carcinoma model due to less responsiveness and resistance of HeLa against cisplatin. Here, attempts are made to investigate the effects of DNA double strand break inhibitor KU-55933 against the cisplatin cell growth and cytotoxicity. Following experiments namely in vitro plasmid DNA metabolizing, Trypan blue dye exclusion, MTT, and PI based Flow cytometery PI assays were conducted to study cell growth and cytotoxicity effects. Based on the cell viability and PI based staining data, results remarked that KU-55933 combined with cisplatin could bring convincing cell growth arrest in HeLa. The reduction in HeLa proliferation was noticed from 70% to 30% in case of KU-55933 added with cisplatin over cisplatin alone. However, we noticed none apoptosis based cell cytotoxicity in case of cisplatin alone or combined with the inhibitors. We also observed significant DNA instability in case of KU-55933 treated HeLa lysates added to plasmid DNA substrate over HeLa lysate without KU-55933 treatment. In conclusion, KU-55933 can potentiate low dose of cisplatin response against HeLa. The effect of KU-55933 may not be attributed due to its enhancing the apoptosis way, rather than through cell growth arrest mechanism due to extensive DNA breaks.

Abstract 2718: Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity

Abstract BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase. Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of DNA damage and misattached chromosomes. Here, we report the characterization of a novel benzylpyrazole lead-structure series inhibiting BUB1 exemplified by BAY-320, a novel, first-in-class small molecule inhibitor of BUB1 kinase. This structure class was initially discovered as a single hit in a high-throughput screen, and resulted in a lead compound by chemical optimization. Benzylpyrazole BAY-320 is highly selective for BUB1 with single digit nanomolar biochemical potency and single-digit micromolar cellular potency (HeLa proliferation assay). Synergistic effects can be observed when BUB1 inhibitor BAY-320 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylpyrazoles allowed for a better understanding the binding mode for rational property design. Further data on structure-activity relationship including pharmacokinetic, drug metabolism and the synthesis of BAY-320 and analogues will be presented. These results validate the benzylpyrazoles as novel selective BUB1 inhibitors and BUB1 as a promising approach for cancer treatment. Citation Format: Marion Hitchcock, Gerhard Siemeister, Hans Briem, Amaury Ernest Fernandez-Montalvan, Simon Holton, Anne Mengel, Ursula Mönning, Michael Brands, Karl Ziegelbauer, Dominik Mumberg, Franz von Nussbaum. Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2718.

Emodin induces apoptosis of human cervical cancer hela cells via intrinsic mitochondrial and extrinsic death receptor pathway

BackgroundEmodin is a natural anthraquinone derivative isolated from the Rheum palmatum L. Aim: The aim of the present study was to investigate the effect of emodin on the apoptosis of the human cervical cancer line HeLa and to identify the mechanisms involved.MethodsRelative cell viability was assessed by MTT assay after treatment with emodin. Cell apoptosis was detected with TUNEL, Hoechst 33342 staining and quantified with flow cytometry using annexin FITC-PI staining.ResultsThe percentage of apoptotic cells was 0.8, 8.2, 22.1, and 43.7%, respectively. The mRNA levels of Caspase-9, -8 and −3 detected by Real-time PCR after treatment with emodin were significantly increased. Emodin increased the protein levels of Cytochome c, Apaf-1, Fas, FasL, and FADD but decreased the protein levels of Pro-caspase-9, Pro-caspase-8 and Pro-caspase-3.ConclusionWe conclude that the emodin inhibited HeLa proliferation by inducing apoptosis through the intrinsic mitochondrial and extrinsic death receptor pathways.

Reducing invasion potential of cervical cancer cells via targeted knockdown of c-Jun.

e22005 Background: Despite recent advent of vaccines for human papillomaviruses (HPV) in cervical cancer and increasing efforts to improve therapy, deaths still average 275,000 annually worldwide, with most women succumbing to recurrent or metastatic disease. The c-Jun oncogene is a subunit of the activating protein-1 (AP-1) transcription factor and is strongly expressed in cervical cancer, regulating the expression of HPV16 and 18 genes. AP-1 plays a major role in cell growth, migration and apoptosis in many cell types. This study examined the role of c-Jun in modulating HeLa proliferation, migration, apoptosis and invasion as well as susceptibility to cisplatin. Methods: Transient knockdown and over-expression of c-Jun was performed in HeLa cervical cancer cells using Dharmacon c-Jun siRNA and Origene Jun expression vector. c-Jun and downstream gene/protein expression was confirmed by western blot and real-time PCR and cells subject to proliferation, in vitro wound and matrigel dual-chamber transwell assays. Flow cytometry was used for analysis of cell cycle and apoptosis. Results: c-Jun protein and mRNA levels were reduced by c-Jun siRNA. c-Jun silencing inhibited cervical cancer cell proliferation. Significantly, c-Jun suppression dramatically reduced HeLa migration and invasion and targeted down-regulation of cyclooxygenase-2 (Cox2), interleukin-6 (IL-6), metalloproteinases (MMP)-1, -9 and -13 as well as HPV18 E6 and E7, genes highly expressed in cervical cancer and associated with metastatic growth. Direct siRNA knockdown of Cox2 in HeLa also reduced MMP1 and MMP9 expression suggesting an intermediary link. In HeLa cells over-expressing c-Jun, cell proliferation was not significantly increased but cell invasiveness was markedly enhanced in parallel with enhanced MMP-1 expression. Modulation of c-Jun expression did not interfere with susceptibility of HeLa cells to apoptosis in the presence of cisplatin. Conclusions: Reduced invasion potential of HeLa cells after c-Jun knockdown suggests a potential target in treatment of metastatic and recurrent cervical cancer. Data suggest a mechanism involving down-regulation of Cox2 and MMP-1 and -9 expression.

배양된 HeLa 세포에서 고농도의 에스트로겐, 프로게스테론 및 타목시펜의 세포증식 억제효과

이 연구는 에스트로겐, 프로게스테론 및 타목시펜의 각각 다른 농도와 처리기간에 따라 배양된 사람 난소유래암세포주인 HeLa 세포의 증식에 미치는 영향을 MTT 분석에 의해 조사하였다. 에스트로겐은 2.5~6일의 처리기간 동안 <TEX>$1{\mu}g$</TEX>/ml의 농도까지는 세포증식에 영향을 주지 않았지만, 더 높은 <TEX>$10{\mu}g$</TEX>/ml의 농도에서는 처리기간의 증가에 따라 점진적으로 현저하게 세포증식을 억제하였다. 또한 <TEX>$10{\mu}g$</TEX>/ml 농도 이상의 프로게스테론을 2.5일 동안 처리할 경우 HeLa 세포의 증식을 현저하게 억제하였으며, 4일 동안 처리시에는 농도-의존성 억제효과를 나타내었다. 그러나, 6일 동안 더 장기간의 프로게스테론 처리는 4일 동안의 처리기간에서 관찰된 세포증식에 대한 농도-의존성 억제효과를 제거하였다. 그리고, 타목시펜은 HeLa 세포주의 증식에 대한 억제효과를 위하여 에스트로겐 보다 더 높은 농도(<TEX>$100{\mu}g$</TEX>/ml)를 필요로 하였다. 이러한 결과는 고농도의 에스트로겐, 프로게스테론 그리고 타목시펜이 HeLa 세포의 증식을 억제할 뿐만 아니라, 농도 및 처리기간 또한 세포증식에 대한 억제효과에 영향을 미칠 수 있음을 제시한다. This study examined the effects of estrogen, progesterone and tamoxifen at different concentrations and treatment periods on proliferation of a human cervical carcinoma cell line, HeLa, in culture, based on MTT assay. Estrogen did not have an effect on the cellular proliferation in concentrations up to <TEX>$1{\mu}g$</TEX>/ml for treatment periods of between 2.5 and 6 days, but significantly inhibited proliferation at a higher concentration of <TEX>$10{\mu}g$</TEX>/ml in a progressive manner with increasing treatment periods. Also, treatment of HeLa with more than <TEX>$10{\mu}g$</TEX>/ml of progesterone for 2.5 days significantly inhibited proliferation and caused a concentration-dependent inhibition with 4 days of treatment. However, longer treatment with progesterone for 6 days abolished the concentration-dependent inhibitory effect on cellular proliferation observed with the 4-day treatment period. Furthermore, tamoxifen required a higher concentration (<TEX>$100{\mu}g$</TEX>/ml) than estrogen to bring about the inhibitory effect on the HeLa proliferation. These results suggest that high concentrations of estrogen, progesterone and tamoxifen may suppress proliferation of HeLa, and both the concentration and the treatment period may also influence their inhibitory effects on cellular proliferation.