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

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

MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"BACKGROUND: Matrix metalloproteinases (MMP) are important enzymes in the tumor microenvironment associated with progression of cervical intraepithelial neoplasia (CIN) toward squamous cell carcinoma (SCC) of the cervix. However, the role of MMPs in the inflammatory process associated with Chlamydia trachomatis infection concomitant with the carcinogenic process driven by HPV has not yet been addressed. In the present study, we analyzed the state of the MMP-9-RECK axis in cervical carcinogenesis. METHODS: The levels of MMP-9 and RECK expression were analyzed by immunocytochemistry in liquid-based cytology samples from 136 women with high-grade cervical lesions (CIN2/CIN3) and cervical SCC diagnosed by LLETZ, and in 196 women without cervical neoplasia or CIN1. Real-time qPCR was performed to analyze expression of MMP-9 and RECK in 15 cervical samples. The presence of HPV-DNA and other genital pathogens was evaluated by PCR. RESULTS: We found a higher expression of MMP-9 [OR, 4.2; 95% confidence interval (CI), 2.2-7.8] and lower expression of RECK (OR, 0.4; 95% CI, 0.2-0.7) in women with CIN2/CIN3/SCC when compared with women from the control group (no neoplasia/CIN1). A statistically significant association was also found between MMP-9/RECK imbalance and infection by alpha-9 HPV and C. trachomatis. The prevalence of C. trachomatis infection was significantly higher in women with high-grade cervical disease (OR, 3.7; 95% CI, 1.3-11.3). CONCLUSIONS: MMP-9/RECK imbalance in cervical smears is significantly associated with high-grade cervical diseases and infection by alpha-9 HPV and C. trachomatis. IMPACT: MMP-9/RECK imbalance during cervical inflammation induced by C. trachomatis might play a role in HPV-mediated cervical carcinogenesis.This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), numbers 2008/03232-1 (to L.L. Villa) and 2012/09746-2 (to M.G. Discacciati and S.S. Maria-Engler) and National Counsel of Technological and Scientific Development pharmaceutical innovation (CNPQ-INCT-if; to S.S. Maria-Engler)

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.FAPESP [2006/50479-7, 2006/60930-8, 2008/58817-4, 2009/54816-6 2010/50157-5]FAPESPCNPqCNPqINCT_if (CNPq)INCT-if CNPqCAPESCAPESPRP-USPPRPUS

HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma

A 2D and 3D melanogenesis model with human primary cells induced by tyrosine

Research on melanogenesis, its regulation in health and disease, and the discovery of new molecules with pigmenting and depigmenting activities use different models. Here we standardize a protocol based on previous ones using primary human melanocytes and keratinocytes in co-cultures, in which melanogenesis was induced under mild conditions by the addition of tyrosine plus ammonium chloride (NH4Cl). The expression of MITF, TYR, TYRP1, and Melan-A as well as melanin content were measured. Furthermore, we extended this study to a reconstructed 3D model. Pigmentation was visually observable and melanosomes were identified by Fontana-Masson staining by the addition of tyrosine plus NH4Cl during the stratification phase. The 2D and 3D protocols proposed here circumvent limitations of previous models, using human primary cells and mild conditions for melanogenesis. These protocols offer a viable, robust, simple, and animal-free investigational option for human skin pigmentation studies and screening tests for new compounds that modulate pigmentation

Propolis Reduces the Expression of Autophagy-Related Proteins in Chondrocytes under Interleukin-1β Stimulus

Background: Osteoarthritis (OA) is a progressive and multifactorial disease that is associated with aging. A number of changes occur in aged cartilage, such as increased oxidative stress, decreased markers of healthy cartilage, and alterations in the autophagy pathway. Propolis extracts contain a mixture of polyphenols and it has been proved that they have high antioxidant capacity and could regulate the autophagic pathway. Our objective was to evaluate the effect of ethanolic extract of propolis (EEP) on chondrocytes that were stimulated with IL-1β. Methods: Rabbit chondrocytes were isolated and stimulated with IL-1β and treated with EEP. We evaluated cell viability, nitric oxide production, healthy cartilage, and OA markers, and the expression of three proteins associated with the autophagy pathway LC3, ATG5, and AKT1. Results: The EEP treatment reduces the expression of LC3, ATG5, and AKT1, reduces the production of nitric oxide, increases the expression of healthy markers, and reduces OA markers. Conclusions: These results suggest that treatment with EEP in chondrocytes that were stimulated with IL-1β has beneficial effects, such as a decrease in the expression of proteins associated with autophagy, MMP13, and production of nitric oxide, and also increased collagen II

Epigenetic signature of differentially methylated genes in cutaneous melanoma

Abstract Background Cutaneous melanoma (CM) is the most aggressive subtype of skin cancer, with increasing incidence over the past several decades. DNA methylation is a key element of several biological processes such as genomic imprinting, cell differentiation and senescence, and deregulation of this mechanism has been implicated in several diseases, including cancer. In order to understand the relationship of DNA methylation in CMs, we searched for an epigenetic signature of cutaneous melanomas by comparing the DNA methylation profiles between tumours and benign melanocytes, the precursor cells of CM. Methods We used 20 primary CMs and three primary cell cultures of melanocytes as a discovery cohort. The tumours mutational background was collected as previously reported. Methylomes were obtained using the HM450K DNA methylation assay, and differential methylation analysis was performed. DNA methylation data of CMs from TCGA were recovered to validate our findings. Results A signature of 514 differentially methylated genes (DMGs) was evident in CMs compared to melanocytes, which was independent of the presence of driver mutations. Pathway analysis of this CM signature revealed an enrichment of proteins involved in the binding of DNA regulatory regions (hypermethylated sites), and related to transmembrane signal transducer activities (hypomethylated sites). The methylation signature was validated in an independent dataset of primary CMs, as well as in lymph node and distant metastases (correlation of DNA methylation level: r > 0,95; Pearson’s test: p < 2.2e-16). Conclusions CMs exhibited a DMGs signature, which was independent of the mutational background and possibly established prior to genetic alterations. This signature provides important insights into how epigenetic deregulation contributes to melanomagenesis in general