Cervical Cell Lines Research Articles

Cytotoxic and Antiradical Activities of Extracts of Rhizopora apiculata (L)

The petroleum ether and methanol crude extracts of Rhizopora apiculata (L) (Rhizoporaceae) were screened for cytotoxicity by MTT bioassay and antioxidant activity using three different methods. The active constituents of the plant Rhizopora apiculata (L) were extracted using petroleum ether and methanol. Both extracts were tested for cytotoxicity by MTT assay at a concentration range of 18.75, 37.5, 100, 150 and 300μg/ml. The percentage cell viability shown in Pet ether extract for human cervical adenocarcinoma cell lines (HeLa), human osteosarcoma cell lines (MG 63) and Breast adenocarcinoma (MC 67) were in the range of 102-91%, 100-84% and 101-78% respectively shows that pet ether extract does not have considerable cytotoxic activity. On the other hand, Petroleum ether and methanol crude extracts of Rhizopora apiculata (L) showed mild antioxidant activity with compared to ascorbic acid.

PTC209, a Specific Inhibitor of BMI1, Promotes Cell Cycle Arrest and Apoptosis in Cervical Cancer Cell Lines.

Aberrant expression of the BMI1 oncogene has been prevalently found in a variety of human cancers, including cervical cancer. Recent studies have shown that PTC209, a specific BMI1 inhibitor, exhibits high potency in inhibiting the growth of colon, breast, oral cancer cells and cancer-initiating cells, indicative of its chemotherapeutic potential. In the current study, we evaluated the inhibitory abilities of PTC209 in cervical cancer cells. Three cervical cell lines, C33A, HeLa, and SiHa were treated with PTC209. The impacts of PTC209 on BMI1 were investigated using quantitative reverse-transcription PCR assay (qRT-PCR) and western blotting; changes in cell viability, cell cycle distribution, and apoptosis were assessed using cell viability testing, colony formation assay and flow cytometry analyses, respectively. PTC209 exhibited considerably high short-term and long-term cytotoxicities in all tested cervical cancer cell lines regardless of their HPV infection status, TP53 and pRb statuses. PTC209 significantly downregulated the expression of BMI1 in cervical cancer cell lines, and such downregulation led to G0/G1 arrest (p<0.05). Moreover, PTC209 drove more cells into apoptosis (p<0.05). PTC209 (BMI1-targeting agents, in general) represents a novel chemotherapeutic agent with potential in cervical cancer therapy.

Morindaquinone, a new bianthraquinone from Morinda coreia roots

Phytochemical investigation of the roots of Morinda coreia led to the isolation of one new bianthraquinone, morindaquinone (1), together with 12 known compounds, soranjidiol (2), rubiadin-1-methyl ether (3), 2-methoxy-1,3,6-trihydroxyanthraquinone (4), 1-hydroxy-2-methylanthraquinone (5), tectoquinone (6), nordamnacanthal (7), damnacanthal (8), 2-formylanthraquinone (9), 3-hydroxy-2-hydroxymethylanthraquinone (10), lucidin-ω-methyl ether (11), scopoletin (12) and (+)-mellein (13). The structures of these compounds were determined on the basis of extensive spectroscopic analyses, as well as by comparison with literature reports. Compound 1 was the first example of bianthraquinone found in the genus Morinda, whereas compound 13 was firstly isolated from this genus. Among them, compounds 2, 7, 8 and 10 exhibited moderate to weak cytotoxicity against human cervical (HeLa), human colon (HT 29) and human breast (MCF-7) cell lines, while compounds 6 and 9 – 11 showed weak anti-acetylcholinesterase activity.

Synthesis of Amino Acid-Naphthoquinones and In Vitro Studies on Cervical and Breast Cell Lines.

We performed an extensive analysis about the reaction conditions of the 1,4-Michael addition of amino acids to 1,4-naphthoquinone and substitution to 2,3-dichloronaphthoquinone, and a complete evaluation of stoichiometry, use of different bases, and the pH influence was performed. We were able to show that microwave-assisted synthesis is the best method for the synthesis of naphthoquinone–amino acid and chloride–naphthoquinone–amino acid derivatives with 79–91% and 78–91% yields, respectively. The cyclic voltammetry profiles showed that both series of naphthoquinone–amino acid derivatives mainly display one quasi-reversible redox reaction process. Interestingly, it was shown that naphthoquinone derivatives possess a selective antitumorigenic activity against cervix cancer cell lines and chloride–naphthoquinone–amino acid derivatives against breast cancer cell lines. Furthermore, the newly synthetized compounds with asparagine–naphthoquinones (3e and 4e) inhibited ~85% of SiHa cell proliferation. These results show promising compounds for specific cervical and breast cancer treatment.

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 &gt;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.

Validating interfacial behaviour of surface-active ionic liquids (SAILs) with computational study integrated with biocidal and cytotoxic assessment

Surface-active ionic liquids (SAILs) belonging to the series of N-alkylmethylimidazolium halides [C8mimX] (X = Br, Cl, and BF4) and [CnmimBr] (n = 10, 12, 14, and 16) were employed to understand the influence of hydrophobicity of alkyl chain length and the chaotropicity of counter-ions of SAILs on the micellization, antimicrobial action and cytotoxicity properties. The micellization phenomenon of SAILs in an aqueous environment was examined employing tensiometry and steady-state fluorescence spectrophotometry. The corresponding interfacial parameters viz., critical micelle concentration (CMC), effectiveness (γCMC), surface pressure (ПCMC), maximum surface excess concentration (Гmax), and the minimum area engaged per molecule (Amin) at the air-water interface were evaluated at 303.15 K. These experimental findings were monitored and geometrically optimized theoretically using Gaussian software to highlight the recent advances in this field of theoretical calculations for putative structure. The simulation descriptors correlated the micellization behavior as a function of hydrophobicity which may contribute to obtaining awareness on their ecological behavior and fate. In addition, the biological screening of all the examined SAILs was undertaken with a combined experimental and theoretical (optimized) method against bacteria and fungus. Results revealed that SAILs with the alkyl chain-length greater than C8- act as a fair antimicrobial agent against the selected microbial strain which is attributed to the enhanced degree of SAILs hydrophobicity. The cytotoxicity of these imidazolium-based SAILs was also assessed on the cervical human cell line (HeLa) using the MTT cell viability assay and the data thus obtained were subjected to statistical analysis.

Reduction in the Viability of Human Cervical Cancer HeLa Cell Line via Indirect Co-culture With Amniotic Fluid-Derived Mesenchymal Stem Cells

Objectives: This experiment was carried out to evaluate the impacts of unmodified human amniotic fluid-derived mesenchymal stromal/stem cells (hAF-MSCs) on the viability of HeLa cells, as well as the impact of these cells on the expression of common proapoptotic and pro-survival genes in tumor cells by establishing an indirect co-culture system. Materials and Methods: To this end, an indirect co-culture system was established, and hAF-MSCs were co-cultured with HeLa cells at a ratio of 1:2 for five days. The cell viability of co-cultured tumor cells was determined after the incubation period. Then, several parameters were examined, including the gene expression of tumor protein 53 (TP53), BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A). Finally, gene regulatory networks were analyzed as well. Results: The results of this study confirmed that the co-culture of hAF-MSCs with HeLa cells could decrease the viability of tumor cells. The reduction of HeLa cell viability was accompanied by an increase in BAX, TP53, and CDKN1A while a decrease in BCL2 gene expression. Eventually, the analysis of the regulatory network revealed that the co-culture of Hela ¬cells with hAF-MSCs activated several transcriptional factors and microRNAs which regulated the expression of these genes. Conclusions: In general, hAF-MSCs exerted the inhibitive effects on the growth of HeLa cells, along with alterations in the expression of common pro-apoptotic and pro-survival genes in a timely and concentration-dependent manner.

LINC00511 is associated with the malignant status and promotes cell proliferation and motility in cervical cancer.

LINC00511 is a newly identified lncRNA that is up-regulated in many types of human cancers and may serve as an oncogenic lncRNA. However, there was no report about the role of LINC00511 in cervical cancer. Therefore, we investigated the clinical value of LINC00511 in cervical cancer patients via analyzing the correlation between LINC00511 expression and clinicopathological features. Moreover, we performed loss-of-function study to estimate the effect of LINC00511 on cervical cancer cell proliferation, migration, and invasion. In our study, we found LINC00511 expression levels were increased in cervical cancer tissues and cell lines compared with adjacent normal tissues and normal cervical epithelial cell line, respectively. High LINC00511 expression was correlated with advanced clinical stage, large tumor size, histological type of adenocarcinoma, and present lymph node metastasis, distant metastasis, and poor overall survival in cervical cancer patients. The in vitro studies indicated that knockdown of LINC00511 inhibited cervical cancer cell proliferation, migration, and invasion. In conclusion, LINC00511 acts as oncogenic lncRNA in cervical cancer, and may be a novel biomarker and potential therapeutic target for cervical cancer patients.

Shp2 expression is upregulated in cervical cancer, and Shp2 contributes to cell growth and migration and reduces sensitivity to cisplatin in cervical cancer cells

Src homology phosphotyrosine phosphatase 2 (Shp2) has been found to be overexpressed in cervical cancer tissues. However, the influence of Shp2 on the biological behavior and sensitivity to cisplatin of cervical cancer cells remains unclear. We aimed to assess Shp2 expression in cervical tissues and cell lines and to detect the influence of Shp2 knockdown and overexpression on the biological behavior and sensitivity to cisplatin in cervical cancer cells. We found that Shp2 expression was significantly upregulated in cervical cancer tissues and cell lines, and Shp2 overexpression was associated with lymph node metastasis and a high human papillomavirus (HPV) DNA load. Shp2 knockdown inhibited cell growth and migration and enhanced sensitivity to cisplatin in the HeLa and SiHa cervical cancer cell lines. In contrast, Shp2 overexpression had the opposite effects. These tumor-promoting effects of Shp2 may be partly related to Akt signaling. In conclusion, Shp2 is involved in the occurrence and development of cervical cancer and may confer cisplatin resistance in cervical cancer. Shp2 blockade may be a new strategy for cervical cancer treatment.

Motor Neuron and Pancreas Homeobox 1 (MNX1) Is Involved in Promoting Squamous Cervical Cancer Proliferation via Regulating Cyclin E.

BackgroundCervical cancer is one of the most lethal gynecologic malignancies worldwide. The objective of this study was to assess the role of MNX1 in cervical cancer and its underlying mechanisms.Material/MethodsThe expression of motor neuron and pancreas homeobox 1 (MNX1) in immortal epithelial cervical cell line ECT, cervical cancer cell HeLa, and SiHa and cervical cancer, as well as in adjacent noncancer tissues, was detected and analyzed. CCK-8 and colony formation assays were performed to evaluate the effects of MNX1 overexpression on cervical cancer cell proliferation. Transwell assay was used to detect migration and invasion after MNX1 knockdown or overexpression. Real-time PCR and Western blotting were used to examine MNX1 and cell cycle regulator expression.ResultsData from our study indicated that MNX1 was upregulated both in cervical cancer cell lines and cervical cancer tissues. The high levels of MNX1 are related to advanced stages and lymph nodes metastasis. The overexpression of MNX1 promoted cervical cancer cells proliferation, migration, and invasion. Moreover, MNX1 upregulated 2 critical cell cycle regulators, CCNE1 and CCNE2.ConclusionsThese findings reveal MNX1 as a novel oncogene of cervical cancer and indicate MNX1 is a promising therapeutic and prognostic biomarker.

Myrciaria tenella (DC.) O. Berg (Myrtaceae) Leaves as a Source of Antioxidant Compounds.

Myrciaria species are widely studied to identify their chemical composition and evaluate their biological activity. Since evidence supporting the potential antioxidant and antiproliferative activity of Myrciaria tenella is lacking, the aim of this work was to evaluate these activities in six different leaf extracts: hexane (CHE), chloroform (CCE), ethanolic (CEE), methanolic (CME), aqueous final (CFAE), and only aqueous (CAE). The presence of phenolic compounds, tannin, saponin, and ursolic acid was determined by thin layer chromatography (TLC). CEE, CME, and CFAE showed in vitro antioxidant activity at the initiation, propagation, and termination stages of oxidative damage. Moreover, no toxicity was observed in the 3T3 non-cancerous cell line. On the other hand, all extracts promoted cell death in the tumor cell lines human cervical adenocarcinoma cell line (HeLa) and human stomach gastric adenocarcinoma cell line (AGS). Based on these results, the effect of CEE on the AGS cell line was analyzed using flow cytometry, and necrosis and late apoptosis were observed. Finally, the Caenorhabditis elegans model showed that CEE was able to reduce the basal reactive oxygen species (ROS) level. Ultra-performance liquid chromatography (UPLC) analysis showed rutin as the major compound in CEE. Therefore, Myrciaria tenella fresh leaves may be potential sources of molecules possessing antioxidant and antiproliferative activities.

Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus

Metabolomics generate a profile of small molecules from plant extracts, which could be directly responsible for bioactivity effects. Using dry-column flash chromatography enabled a rapid and inexpensive method for the very efficient separation of plant extract with a high resolution. This separation method coupled to NMR and FTIR-based metabolomics is applied to identify bioactive natural products. OPLS multivariate analysis method, was used for correlation the chemical composition of the plant extracts, Amphoricarpos autariatus, with the results of cytotoxic activity against Human cervical adenocarcinoma cell line (HeLa) and epithelial lung cancer cell line (A549). In this way, the highest contribution to the cytotoxic activity was recorded for the guaianolide sesquiterpene lactones named amphoricarpolides. The compounds indicated as bioactive after metabolomics analysis were tested, and their cytotoxic activity were confirmed.

Burden and genotype distribution of high-risk Human Papillomavirus infection and cervical cytology abnormalities at selected obstetrics and gynecology clinics of Addis Ababa, Ethiopia

BackgroundHuman papillomavirus is recognized as a major cause of cervical cancer. It is estimated that annually, 7,095 women are diagnosed with cervical cancer and 4,732 die from the disease in Ethiopia. Understanding that the screening practice is very poor and the coverage is very limited, this disease burden is one of the major public health agendas in Ethiopia. This study aimed to assess the burden and genotype distribution of high-risk human papillomavirus (HR HPV) infection and cervical cytology abnormalities at selected obstetrics and gynecology clinics of Addis Ababa, Ethiopia.MethodsAn institutional-based cross-sectional study design was employed from June to October 2015. Cervical samples were collected from 366 participants based on inclusion criteria. HR HPV DNA was analyzed using an Abbott Real-Time PCR system, and cervical cytology screening was performed using the conventional Pap-smear technique. Data were entered in to Epi-data version 13 and analyzed using STATA version 11.ResultsThe overall HR HPV burden and abnormal cytology were 13.7 and 13.1%, respectively. The majority of HR HPV types were other than types 16 and 18. Of the total abnormal cytology results, 81.3% were low-grade squamous intraepithelial lesions (LSILs), and 12.5 and 6.3% were atypical squamous cells of undetermined significance (ASCUS) and high-grade squamous intraepithelial lesions (HSILs), respectively. Residence, occupation, and HIV serostatus were significantly associated with HR HPV infection. Among the variables, age, age at first marriage, and education were the only ones associated with cervical cytology abnormalities. The overall agreement between the real-time PCR and Pap cytology screening methods was 78.96% (Kappa value of 0.12, 95% CI (0.00–0.243), P = 0.01).ConclusionsNon-16/18 HR HPV genotypes represented the largest proportion of HR HPV infections in this study. Women without cervical cytology abnormalities had the highest frequency of HR HPV infection. A large-scale community-based cohort study shall be designed and implemented to further identifying the persistent genotype and assessing the changes in cervical epithelial cell lines.

Synthesis and in vitro anticancer activity of new gemcitabine-nucleoside analogue dimers containing methyltriazole or ester-methyltriazole linker.

Two series of novel gemcitabine-nucleoside analogue dimers were synthesized using the 'click' chemistry approach. In the first series of dimers (21-30), the nucleoside units were connected with a stable methyltriazole 4N-3'(or 5')C linker whereas in the second series (31-40) with a cleavable ester-methyltriazole 4N-3'(or 5')C linker. Dimers 21-40 were evaluated for their cytotoxic activity in five human cancer cell lines such as cervical (HeLa), nasopharyngeal (KB), lung (A549), brain (U87), liver (HepG2) and normal dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Compound 29 comprising two gemcitabine (dFdC) units exhibited the highest activity among dimers 21-30. The activity of compound 29 was higher than that of dFdC in all the studied cancer cell lines. A similar order of activity was observed for compounds 25, 28, and 30. The best activity among all the dimers synthesized was displayed by compound 39, comprising two gemcitabine units with a cleavable linker. The activity of compound 39 was 5 to 9 times higher than that of dFdC, depending on the cell line. In addition, marked cytotoxic activity was shown by compounds 31, 36, 38, and 40.

LncRNA HAND2-AS1 inhibits cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and negative cervical squamous cell carcinoma.

Long non-coding (lnc)RNA HAND2 antisense RNA 1 (HAND2-AS1) exhibited tumor suppression activity in different types of cancer. However, its role in cervical squamous cell carcinoma (CSCC), which is frequently diagnosed among females, has not been elucidated. The current study revealed that lncRNA HAND2-AS1 was downregulated, while Rho-associated protein kinase 1 (ROCK1) was upregulated in serum of human papillomavirus (HPV)-positive and -negative patients with CSCC compared with healthy controls. Correlation analysis revealed that the expression levels of lncRNA HAND2-AS1 were negatively correlated with the expression levels of ROCK1 in HPV-positive and -negative patients with CSCC but not in healthy controls. Downregulation of lncRNA HAND2-AS1 distinguished patients with CSCC from healthy controls. Additionally, lncRNA HAND2-AS1 overexpression led to reduced expression levels of ROCK1 in HPV-positive and -negative human CSCC cell lines but not in normal cervical cell lines. ROCK1 overexpression did not significantly affect the expression of lncRNA HAND2-AS1 in all the cell lines investigated. lncRNA HAND2-AS1 overexpression inhibited, while ROCK1 overexpression promoted the proliferation, migration and invasion of HPV-positive and -negative human CSCC cell lines but not normal cervical cell lines. ROCK1 overexpression attenuated the effects of lncRNA HAND2-AS1 overexpression on cancer cell proliferation, migration and invasion. lncRNA HAND2-AS1 may inhibit cancer cell proliferation, migration and invasion by downregulating ROCK1 in HPV-positive and -negative CSCC.

Synthesis of Pyrrolo[3,4-b]pyridin-5-ones via Multicomponent Reactions and In Vitro-In Silico Studies Against SiHa, HeLa, and CaSki Human Cervical Carcinoma Cell Lines.

A series of 12 polysubstituted pyrrolo[3,4-b]pyridin-5-ones were synthesized via a one-pot cascade process (Ugi–3CR/aza Diels-Alder/N-acylation/decarboxylation/dehydration) and studied in vitro using human epithelial cervical carcinoma SiHa, HeLa, and CaSki cell line cultures. Three compounds of the series exhibited significative cytotoxicity against the three cell lines, with HeLa being the most sensitive one. Then, based on these results, in silico studies by docking techniques were performed using Paclitaxel as a reference and αβ-tubulin as the selected biological target. Worth highlighting is that strong hydrophobic interactions were observed between the three active molecules and the reference drug Paclitaxel, to the αβ-tubulin. In consequence, it was determined that hydrophobic–aromatic moieties of bioactive compounds and Paclitaxel play a key role in making stronger interactions to the ligand–target complex. A quantitative structure activity relationship (QSAR) study revealed that the six membered rings are the most significant molecular frameworks, being present in all proposed models for the in vitro-studied cell lines. Finally, also from the docking interpretation, a ligand-based pharmacophore model is proposed in order to find further potential polyheterocyclic candidates to bind stronger to the αβ-tubulin.

Photodynamic therapy of cervical cancer by eradication of cervical cancer cells and cervical cancer stem cells.

The heterogeneous nature of cancer puts cancer stem cells (CSCs) at the beating heart of the tumour. Because of their inherent characteristics of stemness, CSCs evade putative cancer therapies, resulting in treatment resistance or tumour recurrence after a seemingly successful treatment. To prevent treatment resistance and cancer recurrence, killing the beating heart of the tumour is of utmost importance. This study therefore, sought to determine the effect of Photodynamic Therapy (PDT) in eradicating cervical cancer and cervical CSCs. Cervical CSCs were isolated from a cervical adenocarcinoma cell line, HeLa cells, and grown in liquid medium incubated at 37° C, 5% CO2 and 85% humidity. Increasing doses of AlPcSmix photosensitizer were administered to both the total cell population and the isolated CSCs, and irradiated using 673.2 nm diode laser. Post-irradiation cellular changes were observed using biochemical assays and microscopy to determine the response of both the total cell population and the CSCs. Results showed a dose-dependent response of both cell populations to treatment, by demonstration of significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation. The study suggested that PDT using AlPcSmix is a very effective treatment method for the eradication of cervical cancer cells and cervical CSCs, in vitro.

Design, Synthesis, DNA/HSA Binding, and Cytotoxic Activity of Half-Sandwich Ru(II)-Arene Complexes Containing Triarylamine-Thiosemicarbazone Hybrids.

Organoruthenium complexes are potent alternatives for platinum-based complexes because of their superior anticancer activity. In this investigation, a series of new Ru(II)-arene complexes with triarylamine–thiosemicarbazone hybrid ligands with higher anticancer activity than cisplatin are reported. The molecular structure of the ligands and complexes was confirmed spectroscopically and supported by single-crystal X-ray crystallography. These complexes adopted a three-leg piano stool geometry. All the Ru(II)-arene complexes were systematically investigated for their in vitro cytotoxicity against human cervical (HeLa S3), lung (A549) cancer, and human normal lung (IMR-90) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Interestingly, a pyrrolidine-attached Ru(II)-benzene complex exhibited superior activity against cancer cells with low IC50 values, and colony formation study showed complete inhibition at 5 and 10 μM concentration. Furthermore, morphological changes assessed by acridine orange and propidium iodide staining revealed that the cell death occurred by apoptosis. In addition, the interaction between synthesized Ru(II)-arene complexes and DNA/protein was explored by absorption and emission spectroscopy methods. These synthesized new organoruthenium complexes can be used for developing new metal-based anticancer drugs.

Abstract 1401: Sensitive detection of integrated HPV in carcinoma cells

Abstract It is estimated that 79 million people in the US are currently infected with human papillomavirus (HPV). There are more than 200 different HPV types that are common worldwide. HPV types are categorized as either high risk (HR) or low risk (LR). LR types, such as HPV 6 and HPV 11, can cause genital warts or squamous papillomas of the oral cavity, while persistent infection with HR HPV types can induce cervical, anal, or head and neck cancers. Episomal HPV DNA can be detected in advanced stages of cancer. HPV integration in the host genome drives oncogenesis by the expression of the two major viral oncogenes, E6 and E7, which abrogate cell cycle checkpoints, promote cellular proliferation, and cause progressive genetic instability. Hence, screening for the presence of E6/E7 RNA transcripts expression is considered the gold standard for confirming active HPV infection. Early detection of active HPV is critical for better understanding of prognosis and therapeutics. So far, detection of integrated HPV DNA in tissue specimens has been challenging. A novel, cost-effective, in situ hybridization (ISH) detection assay has been developed using two sets of HPV probes with enhanced signaling, termed hyper probes. The Hyper HPV D probe detects HR HPV DNA and the Hyper HPV DR probe can simultaneously detect DNA and E6/E7 RNA of HR HPV subtypes in cancer cell lines and formalin fixed paraffin embedded (FFPE) biopsy specimens. The probes were tested on cervical carcinoma SiHa cells containing 1-3 copies of integrated DNA of HPV 16 and HPV negative cervical cancer C33A cell line. The Hyper HPV DR probe resulted in 1-3 puncta in more than 90% of SiHa cell nuclei, indicating integrated copies of HPV DNA into the host genome. There was also the additional presence of a diffuse signal which was abolished by pretreating SiHa cells with RNase. These data suggest the diffuse signal corresponds to E6/E7 mRNA expression. No signals were detected in HPV negative C33A cells. When cells were stained with Hyper HPV D probes, only punctate signals in nuclei of SiHa cells was present and no signal was evident in C33A control cells. Additionally, human cervical and anal FFPE biopsies were analyzed. Simultaneous presence of both punctate signals and diffuse signals were detected when using Hyper HPV DR probes in HR HPV infected tissues, while only punctate signal was detected when using HPV D hyper probes. The signal from the hyper probes was significantly stronger when compared to other commercially available HPV detection kits. Moreover, other commercially available kits failed to detect low copy of integrated HPV. In conclusion, these data demonstrate the Hyper HPV probes can reliably detect low copies of HPV DNA integrated in the host genome and the presence of HPV mRNA using ISH in HPV infected cells. Citation Format: Maurizio Mauro, Richard Delle Bovi, Jack Coleman. Sensitive detection of integrated HPV in carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1401.