Inhibition of papillomavirus protein function in cervical cancer cells by intrabody targeting

Papillomaviruses (HPVs) are a major cause of human disease, and are responsible for approximately half a million cases of cervical cancer each year. HPVs also cause genital warts, and are the most common sexually transmitted disease in many countries. Despite their importance, there are currently no specific antivirals that are active against HPVs. Papillomavirus protein function is mediated largely by protein-protein interactions, which are difficult to inhibit using conventional approaches. To circumvent these problems, we have prepared an scFv library, and have used this to isolate high-affinity binding molecules that may stearically hinder the association of E6 with p53 and prevent E6-mediated p53 degradation in cervical cancer cells. One of the molecules isolated from the library (GTE6-1), had an affinity for 16E6 of 60nM, and bound within the first zinc finger of the protein. GTE6-1 was able to associate with non-denatured E6 following expression in mammalian cells and could inhibit E6-mediated p53 degradation in in vitro assays. E6-mediated p53 degradation is essential for the continuous growth of cervical cancer cells caused by HPV16. To examine the potential of GTE6-1 as an inhibitor of E6 function in such cells, the molecule was expressed in scFv, diabody and triabody formats in a number of cell lines that are driven to proliferate by the HPV16 oncogenes E6 and E7, including the cervical cancer cell line SiHa. In contrast to small E6-binding peptides containing the ELLG E6-binding motif, GTE6-1 expression lead to changes in nuclear structure, the appearance of apoptosis markers, and an elevation in the levels of p53. No effects were seen with a control scFv molecule, or when GTE6-1 was expressed in cells that are driven to proliferate by simian virus 40 (SV40) T-antigen. Given the accessibility of HPV-associated lesions to topical therapy, our results suggest that large interfering molecules such as intrabodies may be useful inhibitors of viral protein-protein interactions and be particularly appropriate for the treatment of HPV-associated disease.     

Resveratrol significantly inhibits the occurrence and development of cervical cancer by regulating phospholipid scramblase 1

Cervical cancer (CC) is one of the most common female malignancies, and resveratrol is a polyphenol isolated from the skins of grapes, which has been reported to significantly alter the cellular physiology of tumor cells. However, little is known about the role of phospholipid scramblase 1 (PLSCR1) in pathogenesis of CC. Here, we demonstrated that resveratrol could significantly inhibit both the growth of HeLa cells and expression of PLSCR1. These results suggest that resveratrol-mediated cell growth inhibition can be regulated by PLSCR1.     

A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.     

LncRNA MNX1-AS1 promotes the progression of cervical cancer through activating MAPK pathway

Long noncoding RNAs (lncRNAs) have been discovered as significant regulators in a wide range of human cancers. Among them, lncRNA MNX1-AS1 has been proved to be an oncogene in ovarian cancer and glioblastoma. However, the regulatory mechanism of MNX1-AS1 in cervical cancer remains to be understood. Therefore, this study planned to explore the role of MNX1-AS1 in cervical cancer. In the beginning, we found that the expression of MNX1-AS1 was obviously upregulated in cervical cancer tissues and cell lines. Kaplan-Meier survival analysis revealed that patients with higher MNX1-AS1 expression level suffered from shorter overall survival time than those with lower MNX1-AS1 level. Moreover, by loss-of-function and gain-of-function assay, the effect of MNX1-AS1 on cell proliferation and apoptosis was examined on cellular level. Results showed that the proliferation of Hela cells was significantly inhibited and apoptosis enhanced by the transfection of shMNX1-AS1, while overexpressing MNX1-AS1 in E6E7 cells presented the contrary results. As for mechanism investigation, it was demonstrated that overexpression of MNX1-AS1 significantly improved the expression of p-ERK1/2 and p-JNK. And the effects of MNX1-AS1 on cell proliferation and apoptosis would be diminished after inactivating the phosphorylation of either ERK or JNK. Taken together, it was identified that MNX1-AS1 promoted proliferation and inhibited apoptosis of cervical cancer cells through MAPK pathway.     

Apoptotic effect of eugenol in human colon cancer cell lines

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub-G1-phase. Eugenol treatment resulted in reduction of intracellular non-protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol-induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate-ribose polymerase), p53 and caspase-3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol-induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.     

The role of nucleolar spindle-associated protein 1 in human ovarian cancer

Ovarian cancer (OC) is one of the deadliest malignancies of the female reproductive system. The present study focused on the role of Nucleolar spindle-associated protein 1 (NuSAP1) in OC. Relative expression of NuSAP1 was detected in OC tissues as well as cells. After knocking down NuSAP1 with lentivirus-mediated shRNA and verifying the knockdown efficiency via quantitative real-time polymerase chain reaction and Western blot assays, the cell proliferation, apoptosis, and cell cycle were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and flow cytometry, respectively. Transwell assay was conducted to detect the migration and invasion of OC cells. It was showed that NuSAP1 was abundantly expressed in OC tissues and cell lines. After knocking down NuSAP1 in OC cells, in addition to significantly inhibiting proliferation and colony forming ability, it also promotes apoptosis and affects cell cycle distribution. Moreover, cells in the shNuSAP1 group showed significantly suppressed migration and invasion ability compared with that in the shCtrl group. In conclusion, NuSAP1 may act as an oncogenic factor in OC and therefore might serve as an indicator for prognosis and therapeutic target for OC treatment.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

Activation of apoptotic signalling in endothelial cells contributes to the detrimental effects of a variety of pathological stimuli. In investigating the molecular events underlying the anti‐apoptotic effect of human plasma in cultured human endothelial cells, we unexpectedly uncovered a novel mechanism of apoptosis suppression by human plasma through an interaction between two previously unrelated proteins. Human plasma inhibited hypoxia–serum deprivation‐induced apoptosis and stimulated BAD^S136 and Akt^S473 phosphorylation. Akt1 silencing reversed part (~52%) of the anti‐apoptotic effect of human plasma, suggesting the existence of additional mechanisms mediating the anti‐apoptotic effect other than Akt signalling. Human plasma disrupted the interaction of BAD with protein phosphatase 1 (PP1). Mass spectrometry identified fourteen PP1‐interacting proteins induced by human plasma. Notably, a group of serine protease inhibitors including plasminogen activator inhibitor 1 (PAI1), a major inhibitor of fibrinolysis, were involved. Silencing of PAI1 attenuated the anti‐apoptotic effect of human plasma. Furthermore, combined Akt1 and PAI1 silencing attenuated the majority of the anti‐apoptotic effect of human plasma. We conclude that human plasma protects against endothelial cell apoptosis through sustained BAD phosphorylation, which is achieved by, at least in part, a novel interaction between PP1 with PAI1.     

CLEFMA induces intrinsic and extrinsic apoptotic pathways through ERK1/2 and p38 signalling in uterine cervical cancer cells

Although concurrent chemoradiotherapy is the cornerstone of treatment for locally advanced or recurrent uterine cervical cancer, treatment fails at a high rate. Therefore, the development of novel targeting agents is critical. This study investigated the action of CLEFMA, a potent, synthetic curcumin derivative, on cervical cancer cells and its mechanism of action. We found that CLEFMA negatively regulated the viability of cervical cancer cells, involving induction of cell apoptosis. Cleaved caspase-3, cleaved poly(adenosine diphosphate-ribose) polymerase, cleaved caspase-8, and cleaved caspase-9 expression were increased by treatment with CLEFMA. After U0126 (ERK1/2 inhibitor) and SB203580 (p38 inhibitor) were applied as cotreatment with CLEFMA, the expression of cleaved caspase-8, -9, and -3 was reduced significantly. In conclusion, CLEFMA activates both extrinsic and intrinsic apoptotic pathways through ERK1/2 and p38 signal transduction in cervical cancer cells.     

Celastrol inhibit the proliferation,invasion and migration of human cervical HeLa cancer cells through down-regulation of MMP-2 and MMP-9

The present study evaluated the anticancer potential of celastrol through down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP-2, MMP-9 and caspase-3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1-100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol-treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP-2 and MMP-9 and increased expression of caspase-3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.     

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.     

白藜芦醇通过诱导细胞自噬性死亡抑制宫颈癌的研究

白藜芦醇作为一种广泛存在于药食同源植物中的非黄酮类多酚化合物,其抗肿瘤效果受到广泛关注,但在抑制宫颈癌方面仍缺乏体内效应的实验依据.本研究通过体内实验发现白藜芦醇具有明显的抗肿瘤生长作用,组织水平LC3B、P62和Beclin-1表达改变,推测白藜芦醇可能通过促进癌细胞的过度自噬抑制宫颈癌的进展;进一步通过体外细胞实验...