Cervical Cancer Cell Lines ME-180 Research Articles

Effects of curcumin photodynamic therapy on notch signaling pathway in cervical cancer cells

Objective To explore the mechanism of notch signaling pathway in the treatment of cervical cancer with notch receptor blocker(DAPT). Methods After treatment of Me180 cells with different concentrations of DAPT combined with curcumin-photodynamic therapy(PDT), 3-(4, 5 Dimethylthiazlo-yl)-2, 5 Dimethylthiazol-2-yl)-2, 5 dipheny tetrazo lium bromide(MTT)assay was used to detect the effect on the proliferation of Me180 cells, annexin V-FITC/PI combined with flow cytometry was used to detect the effect on apoptosis, and real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of Notch1 gene respectively. Results Both DAPT(1.0 μmol/L)and curcumin-PDT groups could inhibit the proliferation and induce apoptosis of cervical cancer cell line Me180, and the synergistic effect of the two groups was significant(P<0.05 or<0.01). DAPT and curcumin-PDT group reduced the mRNA expression level of Notch1 in cervical cancer Me180 cells with inhibition ratio of 40.0% and 32.3% respectively.DAPT and curcumin-PDT group inhibited the protein expressions of Notch1, nuclear factor-kappa B(NF-kB), vascular endothelial growth factor(VEGF), and the synergistic effect of the two groups was statistically significant. Conclusions Both DAPT and curcumin-PDT may effectively block the conduction of Notch signaling pathway, which is associated with down-regulation of the expression of Notch1 and NF-kappa B. Notch signaling pathway may be one of the targets of curcumin photodynamic therapy. Key words: Curcumin; Photodynamic therapy; Apoptosis; Uterine cervical neoplasms

Abstract 2803: Distinct roles of intracellular ROS in cisplatin and evofosfamide cytotoxicity

Abstract Cisplatin is one of the most widely used anticancer agents for the treatment of an array of malignancies. Although multiple mechanisms are involved in cisplatin efficacy, the most prominent and best understood modes of action are the generation of DNA lesions and induction of apoptosis and mitochondrial reactive oxygen species (ROS)-dependent effects on mitochondrial integrity. However, intrinsic or acquired cisplatin resistance can become a limiting factor in cisplatin-based therapy. Evofosfamide is a hypoxia-activated prodrug that is reduced at its nitroimidazole group and preferentially under hypoxic conditions releases Br-IPM, a DNA cross-linker. Evofosfamide is currently being investigated in multiple clinical trials for the treatment of cancer including indications and settings where resistance to cisplatin is observed (e.g. second-line non-small cell lung cancer; NCT01403610). We have previously demonstrated both shared and distinct DNA repair pathways involved with evofosfamide and cisplatin DNA damage. In the current study, we have conducted comparative studies to identify any distinctions in the relative role of ROS underlying cell sensitivity to cisplatin and evofosfamide to help evaluate the potential of evofosfamide to treat cisplatin-resistant tumors. The cisplatin-sensitive cervical cancer cell line ME180, the intermediate cisplatin-resistant human ovarian cancer cell line SK-OV-3, and cisplatin-resistant human lung cancer cell line A549 were investigated with respect to their chemosensitivity to evofosfamide. Unlike cisplatin, evofosfamide exhibited similar potency across all three cell lines. Addition of the ROS scavenger N-acetyl-L-cysteine (NAC) reduced the potency of cisplatin- but not evofosfamide-mediated cytotoxicity, indicating ROS plays a role in cytotoxicity mediated by cisplatin, but not evofosfamide. ROS levels were investigated using the dye H2DCFDA. Cisplatin induced a concentration-dependent and NAC-inhibitable increase of cellular ROS in cisplatin-sensitive ME-180 cells but not in cisplatin-resistant A549 cells. In contrast, ROS levels were not affected by the addition of evofosfamide under normoxia and hypoxia in both ME180 and A549 cells. In order to ascertain whether mitochondrial pathway-mediated apoptosis was involved in cytotoxicity mediated by evofosfamide and cisplatin, mitochondrial membrane depolarization was investigated with the dye JC-1. Cisplatin induced a concentration-dependent JC-1 polarization and these effects were abolished by NAC. In contrast, evofosfamide did not affect mitochondrial membrane potential. In conclusion, this data demonstrates that ROS is an essential factor in cisplatin-mediated cytotoxicity, but not in evofosfamide-mediated cytotoxicity. Citation Format: Fanying Meng, Deepthi Bhupathi, Geraldine Chan, Charles P. Hart. Distinct roles of intracellular ROS in cisplatin and evofosfamide cytotoxicity. [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 2803.

Facile Diversity-Oriented Synthesis of Polycyclic Pyridines and Their Cytotoxicity Effects in Human Cancer Cell Lines.

A three-component cascade method has been developed for the direct synthesis of polysubstituted pyridines. This strategy provides a very convenient route to pyridines using a variety of β-bromo-α,β-unsaturated aldehydes, 1,3-diketones, and ammonium acetate without any additional catalyst or metal salt under mild conditions. A variety of β-ketoesters and 4-hydroxycoumarin were also used instead of 1,3-diketones for the diverse synthesis of polycyclic pyridines. One of the synthesized pyridines has been unambiguously established by a single crystal XRD study. All of the synthesized pyridine derivatives were evaluated for their antiproliferative properties in vitro against the human cancer cell lines HeLa, Me180, and ZR751. Compounds 4{4,1} and 4{2,4} showed significant cytotoxicity in the human breast cancer cell line ZR751 and cervical cancer cell line Me180, respectively, and a few other compounds were found to have moderate activities.

Synthesis and anticancer potential of benzothiazole linked phenylpyridopyrimidinones and their diones as mitochondrial apoptotic inducers

A series of benzothiazole linked phenylpyridopyrimidinones (8a–g) and their diones (9a–g) have been designed, synthesized and evaluated for their anticancer activity. Among the series one of the conjugate 8b showed significant cytotoxicity against human cervical cancer cell line ME-180 with IC50 value of 4.01μM. This compound was tested on the cell cycle perturbations and DNA damage. Flow cytometry analysis revealed that the compound 8b showed drastic cell cycle perturbations due to concentration dependent increase in the sub-G0 phase in ME-180 cell line. DNA fragmentation and Hoechst staining reveals that this compound induced cell death by apoptosis. Further caspase-3 and loss of mitochondrial membrane potential suggested that the compound induces cell death by apoptosis.

A study on the mechanism of 5-aminolevulinic acid photodynamic therapy in vitro and in vivo in cervical cancer

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is a new ablation treatment for tumors, while its function mechanism in cervical cancer has not been elucidated. In this study, we investigated the effects of ALA-PDT on the cervical cancer cell line Me180, to search for optimal parameters of PDT. ALA-PDT on proliferation of Me180 was examined by MTT assay to find the optimal function parameters of ALA-PDT. Apoptosis was observed by using AnnexinV-FITC/PI double staining, Hoechst 33342 staining and May-Grünwald-Farbstoff Giemsa staining. Furthermore, we established a tumor model and 6 mice of each group underwent measurement of the tumor size on days 3, 7, 14, and 21 after treatment. The mRNA expression of survivin, bcl-2, p53, bax and bad in Me180 cells were detected by real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) in vitro and in vivo. Finally, we compared the effects between topical and intravenous administration. Based on the above studies, we found ALA-PDT induced apoptosis and G0-G1 phase arrest of Me180 cells. The tumor volume of the topical administration and PDT group was the smallest at 7-14 days post-PDT. H&E staining showed remarkable subcutaneous necrosis in the PDT groups. The mRNA expression of survivin and bcl-2 in Me180 cells were suppressed post-PDT. Topical administration of PDT is recommended in treating cervical cancer so as to minimize the side-effects and inconvenience of phototoxic reaction brought by PDT. Our data may contribute to making the mechanism of PDT on cervical cancer clearer and give some useful suggestions for clinical application.

Cervical Cancer Cells Present HPV-E7 Epitope to CD4+ Tumor Infiltrating Lymphocytes (TIL) in Association with Histocompatibility Leukocyte Antigen (HLA) DR4 - Identification of HPV-peptides by CD4+ T-cells -

Purpose: Cellular and humoral immune responses directed against autologous tumor cells in patients with cervical cancer may be directed against proteins provided by human papilloma- virus (HPV) associated products or, alternatively, by yet undefined targets as well. The goal of our study was to evaluate the local cellular immune response in cervical cancer. Methods: From a fresh tumor sample of a patient with cervical carcinoma, tumor-infiltrating lymphocytes (TIL) were generated, expanded and characterized by immunohistochemistry, flow cytometry, cytokine release assays and DNA fragment analysis. Results: We established a MHC class II-restricted CD4+ T-cell line from a patient with cervical cancer which recognizes autologous (HPV35+, HPV59+) tumor cells and the HLA-DR4-matched cervical cancer cell line Me180 (HPV68+) as determined by TNFα secretion. Expression of different HPV-E7 genes in autologous B-cells revealed that this T-cell line defines a DR4-presented T-cell epitope which is shared among the E7 gene of HPV59 and HPV68. Conclusion: Tumor-HPV-specific and MHC-class II-restricted CD4+ T-cells are present within the tumor lesion and can be successfully expanded in the presence of IL-2 and IL-7. MHC class II presented peptides may be implemented to augment T-cell responses directed against autologous tumor cells.

CD4+ Tumor-Infiltrating Lymphocytes in Cervical Cancer Recognize HLA-DR-Restricted Peptides Provided by Human Papillomavirus-E7

Human papillomavirus (HPV)-encoded proteins may provide targets for CD8+ or CD4+ T lymphocytes infiltrating into cervical cancer. We established an MHC class II-restricted CD4+ T cell line from a patient with cervical cancer that recognizes autologous (HPV35+, HPV59+) cervical cancer cells and the HLA-DR4-matched cervical cancer cell line Me180 (HPV68+) as determined by TNF-alpha secretion. Expression of different HPV-E7 genes in autologous B cells revealed that this T cell line defines a DR4-presented T cell epitope that is shared among the E7 genes of HPV59 and HPV68. MHC class II-presented peptides may be implemented to augment T cell responses directed against autologous tumor cells, particularly if cancer cells lack MHC class I expression, which is a frequent event in the evolution of cervical cancer.

Interferon-α and All-trans-Retinoic Acid Reversibly Inhibit thein VitroProliferation of Cell Lines Derived from Cervical Cancers

Interferon and retinoic acid are active agents for the treatment of cervical cancer, but their mechanisms of action are unclear. Results of [3H]thymidine uptake assays showed that exposure to pharmacologic concentrations of interferon-α (IFN-α) and all-trans-retinoic acid (RA) for 72 hr inhibited growth of the cervical cancer cell lines ME-180, 283, SiHa, C33-A, 621, CaSki, HeLa, and B132. CaSki and SiHa cells continuously exposed to IFN-α or RA or both for 9 days developed resistance to growth inhibition, and growth resumed at a rate comparable to control after removal of agents. Similar assays showed no significant difference in effects of RA and itscisisomer. Assays for lactate dehydrogenase release revealed no significant lysis of any cell line following exposure to IFN-α, RA, or their combination. In organotypic culture, cells grew in a pattern histologically similar to carcinomain situ,and exposure to IFN-α and RA for 14 days yielded no change in this pattern. Immunohistochemical analysis showed no change in cytokeratin expression by cells in organotypic or monolayer culture. The majorin vitroeffect of IFN-α and RA on cervical cancer cell lines appears to be reversible inhibition of proliferation.

Effects of scheduling and ascites-associated macrophages on combined antiproliferative activity of alpha-2b interferon and gamma-interferon in a clonogenic assay.

Effects of combination treatment with human recombinant alpha-2b interferon (IFN-alpha 2b) and gamma interferon (IFN-gamma) and sequencing of the combination on colony formation of human tumor cells were studied in a human tumor clonogenic assay (HTCA) with or without ascites-associated macrophages (AAM). Five different human tumor cell lines were studied. Three of the five cell lines (ovarian cancer cell line BG-1, cervical cancer cell line ME-180, and melanoma cell line SK-MEL 28) were sensitive to both IFNs. Cervical cancer cell line CaSki was sensitive to IFN-alpha 2b but resistant to IFN-gamma. Endometrial cancer cell line HEC-1A was resistant to both IFNs. Synergistic interaction was observed in BG-1 and SK-MEL 28 with a combination of the IFNs. ME-180 did not exhibit a positive interaction, in spite of its sensitivity to each IFN. CaSki and HEC-1A also did not exhibit a positive combined interaction at clinically achievable concentrations. One sequential combination method (method 1: IFN-alpha 2b----IFN gamma with a 24-h interval) resulted in a similar antitumor effect as the simultaneous combination. A reversed sequential method (method 2: IFN-gamma----IFN-alpha 2b with a 24-h interval) was less effective in three of the five cell lines. In BG-1, AAM enclosed in the lower layer markedly enhanced the antitumor effect of combined IFNs as well as each IFN alone. The antitumor effect with method 1 was significantly greater than that achieved with simultaneous combination or combination according to method 2 in the presence of AAM (P less than 0.01). These results suggest that (1) a synergistic antitumor effect of IFN-alpha 2b and IFN gamma is demonstrable in selected types of tumors, depending upon the sensitivity of each tumor cell line to both IFNs; (2) optimal scheduling for the direct antitumor effect of combined IFNs seems to be long-term exposure of cells to the IFN, the cells being treated with both IFNs either simultaneously or sequentially (IFN-alpha 2b preceding IFN-gamma); and (3) AAM potentiate the antitumor effect of IFNs either alone or in combination. Finally, IFN-alpha 2b may have some priming effects for the indirect effect of IFN gamma mediated through AAM in certain tumor cells.