Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Retinoids: Molecular Aspects and Treatment in Premalignant Lesions and Cervical Cancer.

Persistent infection with high-risk human papillomavirus remains the primary factor associated with the progression of cervical squamous intraepithelial lesions and the development of cervical cancer. Nevertheless, a combination of factors, including genetic predisposition, immune response, hormonal influences, and nutritional status, contribute synergistically to the development of cervical cancer. Among the various factors involved in the pathogenesis and therapy of cervical cancer, retinoids have gained considerable attention due to their multifaceted roles in different cellular processes. This review investigates defects within the vitamin A metabolism pathway and their correlation with cervical cancer. Additionally, it integrates epidemiological and experimental findings to discuss the potential utility of retinoid-based therapies, either alone or combined with other therapies, as agents against premalignant lesions and cervical cancer.

The vaginal and fecal microbiota of a murine cervical carcinoma model under synergistic effect of 17ß-Estradiol and E7 oncogene expression.

Cervical cancer is an important health issue worldwide. Many factors are related to this condition as the persistence of human papillomavirus (HPV) infection (e.g. type 16 and 18), the use of hormonal contraceptives for long periods of time, pH changes and bacterial vaginosis. The association between the microbiota and cervical human cancer is an interesting issue to be explored; given that environmental and hormonal factors may change the vaginal microbiota contributing to this condition. Our hypothesis was that changes in the microbiota diversity is associated with the development of cervical cancer. We evaluated the microbiota diversity in vaginal lavages and fecal samples at different stages of cervical cancer development in a mice model (K14HPV16E7) with type 16 E7 oncogene expression (E7), under continuous or not continuous stimulus of 17ß-estradiol (E2) and compared it with a non-transgenic isogenic control (FVB) under same conditions. Our results indicate that continuous E2 administration during 6 months in the model with type 16 E7 expression causing development of cancer, is associated with significant changes in the microbiota diversity of the cervicovaginal lavages. Similar results were not observed in the same model when no E2 was administered to the mice. The FVB mice with no E7 expression which do not develop cervical cancer, did not show comparable changes in the microbiota diversity when E2 was administered during the same period. Normal evolution of the cervical epithelium and microbiota diversity were observed for the FVB mice with no E2 administration. Large changes in the microbiota diversity in fecal samples were not observed suggesting a specific organ effect of E7 expression associated to E2 on the vaginal microbiota.

Mutant p53R248Q downregulates oxidative phosphorylation and upregulates glycolysis under normoxia and hypoxia in human cervix cancer cells.

Mutations in p53 are strongly associated with several highly malignant cancer phenotypes but its role in regulating energy metabolism has not been completely elucidated. The effect on glycolysis and oxidative phosphorylation (OxPhos) of mutant p53R248Q overexpression in HeLa cells (HeLa-M) was analyzed and compared with cells overexpressing wild-type p53 (HeLa-H) and nontransfected cells containing negligible p53 levels (HeLa-L). p53 R248Q overexpression induced early cell detachment during in vitro growth; however, detached HeLa-M cells showed high viability, shorter generation time and significant diminution in the adhesion proteins E-cadherin and ß-catenin versus HeLa-H and HeLa-L cells. Under normoxia, a lower growth rate of attached HeLa-M cells correlated with decreased levels of proliferating cell nuclear antigen (PCNA), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), mitochondrial proteins (20-80%) and OxPhos flux (69 ± 12%). On the contrary, HeLa-M also showed increased contents of CDKN1A, nuclear factor κB (NF-κB), c-MYC, hypoxia-inducible factor 1-α (HIF-1α; 1-4 times), glycolytic HIF-1α targets (2-4 times), and glycolysis flux (2-fold) versus HeLa-H. In consequence, glycolysis provided ~70% of the cellular adenosine triphosphate (ATP) in HeLa-M cells under normoxia whereas, OxPhos predominated (65-82%) in HeLa-H and HeLa-L cells. Pifithrin-α, a specific p53 inhibitor, did not alter the p53 R248Q target protein contents and OxPhos and glycolytic fluxes, and a poor HIF-1α-p53 R248Q interaction was attained, in HeLa-M cells. These observations suggested that p53 R248Q deficiently interacted with pifithrin-α and HIF-1α. Therefore, lower mitochondrial biogenesis, deficient HIF-1α/mutant p53 interaction, and development of a pseudohypoxic state under normoxia were the apparent biochemical mechanisms underlying glycolysis activation and OxPhos downregulation in HeLa-M cells.

MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

Dual regulation of energy metabolism by p53 in human cervix and breast cancer cells.

The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2-5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2level.

Shorter telomeres and high telomerase activity correlate with a highly aggressive phenotype in breast cancer cell lines.

Maintenance of telomere length is one function of human telomerase that is crucial for the survival of cancer cells and cancer progression. Both telomeres and telomerase have been proposed as possible biomarkers of cancer risk and cancer invasiveness; however, their clinical relevance is still under discussion. In order to improve our understanding of the relationship between telomere length and telomerase activity with cancer invasiveness, we studied telomere length as well as telomerase levels, activity, and intracellular localization in breast cancer cell lines with diverse invasive phenotypes. We found an apparently paradoxical coincidence of short telomeres and enhanced telomerase activity in the most invasive breast cancer cell lines. We also observed that hTERT intracellular localization could be correlated with its level of activity. There was no association between human telomerase reverse transcriptase (hTERT) protein expression levels and invasiveness. We propose that simultaneous evaluation of these two biomarkers-telomere length and telomerase activity-could be useful for the assessment of the invasive capacity and aggressiveness of tumor cells from breast cancer patients.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

Astemizole-based anticancer therapy for hepatocellular carcinoma (HCC), and Eag1 channels as potential early-stage markers of HCC.

Hepatocellular carcinoma (HCC) has very poor prognosis. Astemizole has gained great interest as a potential anticancer drug because it targets several proteins involved in cancer including the Eag1 (ether à-go-go-1) potassium channel that is overexpressed in human HCC. Eag1 channels are regulated by cancer etiological factors and have been proposed as early tumor markers. Here, we found that HepG2 and HuH-7 HCC cells displayed Eag1 messenger RNA (mRNA) and protein expression, determined by real-time RT-PCR and immunochemistry, respectively. Astemizole inhibited human HCC cell proliferation (assessed by metabolic activity assay) and induced apoptosis (studied with flow cytometry) in both cell lines. The subcellular Eag1 protein localization was modified by astemizole in the HepG2 cells. The treatment with astemizole prevented diethylnitrosamine (DEN)-induced rat HCC development in vivo (followed by studying γ-glutamyl transpeptidase (GGT) activity). The Eag1 mRNA and protein levels were increased in most DEN-treated groups but decreased after astemizole treatment. GGT activity was decreased by astemizole. The Eag1 protein was detected in cirrhotic and dysplastic rat livers. Astemizole might have clinical utility for HCC prevention and treatment, and Eag1 channels may be potential early HCC biomarkers. These data provide significant basis to include astemizole in HCC clinical trials.

The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFß pathway in the epidermis of the K14E6 transgenic mouse.

High-risk human papillomaviruses (HR-HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non-melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor ß (TGFß) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFß pathway in the epidermis of K14E6 mice. We used 8-day-old K14E6 and non-transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFß pathway in epidermis of K14E6 mice by downregulation of the TGFß type II receptor (TßRII). This loss of TßRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFß signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation.

Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein.

Ouabain induces endocytosis and degradation of tight junction proteins through ERK1/2-dependent pathways.

In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.

Embryonic stem cell-specific signature in cervical cancer.

The wide range of invasive and noninvasive lesion phenotypes associated with high-risk human papillomavirus (HR-HPV) infection in cervical cancer (CC) indicates that not only the virus but also specific cervical epithelial cells in the transformation zone (TZ), such as stem cells (SCs), play an important part in the development of cervical neoplasia. In this review, we focused in an expression signature that is specific to embryonic SCs and to poorly differentiated cervical malignant tumors and we hypothesize that this expression signature may play an important role to promote cell growth, survival, colony formation, lack of adhesion, as well as cell invasion and migration in CC.

Papillomavirus E6 oncoprotein up-regulates occludin and ZO-2 expression in ovariectomized mice epidermis.

We have studied the expression of the tight junction proteins (TJ) occludin, claudin-1 and ZO-2 in the epidermis of female mice. We observed a peak of expression of these proteins at postnatal day 7 and a decrease in 6 week-old mice to values similar to those found in newborn animals. We explored if the expression of the E6 oncoprotein from high-risk human papilloma virus type 16 (HPV16) in the skin of transgenic female mice (K14E6), altered TJ protein expression in a manner sensitive to ovarian hormones. We observed that in ovariectomized mice E6 up-regulates the expression of occludin and ZO-2 in the epidermis and that this effect was canceled by 17ß-estradiol. Progesterone instead induced occludin and ZO-2 over-expression. However, the decreased expression of occludin and ZO-2 induced by 17ß-estradiol in the epidermis was not overturned by E6 or progesterone. In addition, we employed MDCK cells transfected with E6, and observed that ZO-2 delocalizes from TJs and accumulates in the cell nuclei due to a decrease in the turnover rate of the protein. These results reinforce the view of 17ß-estradiol and E6 as risk factors for the development of cancer through effects on expression and mislocalization of TJ proteins.

Antiproliferative and proapoptotic effects of astemizole on cervical cancer cells.

OBJECTIVE: Cervical cancer is a major cause of mortality among women in developing countries. Thus, it is necessary to offer novel therapies to treat this malignancy. Astemizole has been suggested as a novel and interesting anticancer agent because it targets several proteins involved in cancer including Eag1 (ether à-go-go-1) potassium channels. Eag1 has been proposed as a tumor marker for different types of cancer. Actually, we previously suggested Eag1 channels as cervical cancer and dysplasia markers. Besides, Eag1 has been proposed as a therapeutic target for different malignancies. However, the effect of astemizole in cervical cancer cells is unknown. Therefore, we investigated the effect of astemizole on the proliferation and apoptosis of cervical cancer cells. METHODS: Five cervical cancer cell lines (HeLa, SiHa, CaSki, INBL, and C-33A) were cultured according to manufacturer's instructions. Eag1 protein expression was studied by immunocytochemistry. Cell proliferation was assayed with the MTT method, and apoptosis was investigated by flow cytometry. RESULTS: Eag1 protein expression was observed in different cell lines. Astemizole decreased cell proliferation in up to 40% and increased apoptosis severalfold in all the cell lines studied. CONCLUSIONS: Our results suggest astemizole as a potential therapy for cervical cancer.

The role of Lin28A and Lin28B in cancer beyond Let-7.

Lin28A and Lin28B are paralogous RNA-binding proteins that play fundamental roles in development and cancer by regulating the microRNA family of tumor suppressor Let-7. Although Lin28A and Lin28B share some functional similarities with Let-7 inhibitors, they also have distinct expression patterns and biological functions. Increasing evidence indicates that Lin28A and Lin28B differentially impact cancer stem cell properties, epithelial-mesenchymal transition, metabolic reprogramming, and other hallmarks of cancer. Therefore, it is important to understand the overexpression of Lin28A and Lin28B paralogs in specific cancer contexts. In this review, we summarize the main similarities and differences between Lin28A and Lin28B, their implications in different cellular processes, and their role in different types of cancer. In addition, we provide evidence of other specific targets of each lin28 paralog, as well as the lncRNAs and miRNAs that promote or inhibit its expression, and how this impacts cancer development and progression.

Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells.

Lin28A is an oncoprotein overexpressed in several cancer types such as testicular, ovarian, colon, breast and lung cancers. As a pluripotency factor that promotes tumorigenesis, Lin28A is associated with more undifferentiated and aggressive tumors phenotypes. Moreover, Lin28A is a highly stable protein that is difficult to downregulate. The compound resveratrol (RSV) has anticancer effects. The present study aimed to elucidate the mechanisms underlying the downregulation of Lin28A protein expression by RSV in the NCCIT cell line. NCCIT cells were treated with different concentrations of RSV to investigate its effects on Lin28A expression. The mRNA expression levels of Lin28A and ubiquitin-specific protease 28 (USP28) were assessed using reverse transcription-quantitative PCR. Western blot analysis was employed to evaluate the protein levels of Lin28A, USP28 and phosphorylated Lin28A. In addition, in some experiments, cells were treated with a MAPK/ERK pathway inhibitor, and other experiments involved transfecting cells with small interfering RNAs targeting USP28. The results demonstrated that RSV significantly reduced Lin28A expression by destabilizing the protein; this effect was mediated by the ability of RSV to suppress the expression of USP28, a deubiquitinase that normally protects Lin28A from ubiquitination and degradation. Additionally, RSV inhibited phosphorylation of Lin28A via the MAPK/ERK pathway; this phosphorylation event has previously been shown to enhance the stability of Lin28A by increasing its half-life. This resulted in Lin28A degradation through the proteasomal pathway in NCCIT cells. The results provide further evidence of the anticancer activity of RSV, and identified Lin28A and USP28 as promising therapeutic targets. As a stable oncoprotein, downregulating Lin28A expression is challenging. However, the present study demonstrated that RSV can overcome this hurdle by inhibiting USP28 expression and MAPK/ERK signaling to promote Lin28A degradation. Furthermore, elucidating these mechanisms provides avenues for developing targeted cancer therapies.

Role of SLC5A8 as a Tumor Suppressor in Cervical Cancer.

BACKGROUND: The SLC5A8 gene is silenced in various types of cancer, including cervical cancer; we recently demonstrated that the SLC5A8 gene is also silenced in cervical cancer by hypermethylation of the CpG island in the gene promoter. This study aims to analyze whether SLC5A8 could be a tumor suppressor in cervical cancer. METHODS: After ectopic expressing SLC5A8 in the HeLa cell line, we evaluated its effects on cell behavior both in vitro and in vivo by Confocal immunofluorescence, cell proliferation, migration assays, and xenograft transplants. RESULTS: Overexpression of SLC5A8 in the HeLa cell line decreased its proliferation by arresting cancer cells in the G1 phase and inhibiting cellular migration. Furthermore, we observed that pyruvate increased the SLC5A8 effect, inducing S-phase arrest and inhibiting the entry into mitosis. SLC5A8 decreased tumor growth in xenograft transplants, significantly reducing the volume and tumor weight at 35 days of analysis. CONCLUSIONS: In summary, our results indicate that SLC5A8 has a role as a tumor suppressor in cervical cancer.

In situ molecular identification of the influenza A (H1N1) 2009 Neuraminidase in patients with severe and fatal infections during a pandemic in Mexico City.

BACKGROUND: In April 2009, public health surveillance detected an increased number of influenza-like illnesses in Mexico City's hospitals. The etiological agent was subsequently determined to be a spread of a worldwide novel influenza A (H1N1) triple reassortant. The purpose of the present study was to demonstrate that molecular detection of pandemic influenza A (H1N1) 2009 strains is possible in archival material such as paraffin-embedded lung samples. METHODS: In order to detect A (H1N1) virus sequences in archived biological samples, eight paraffin-embedded lung samples from patients who died of pneumonia and respiratory failure were tested for influenza A (H1N1) Neuraminidase (NA) RNA using in situ RT-PCR. RESULTS: We detected NA transcripts in 100% of the previously diagnosed A (H1N1)-positive samples as a cytoplasmic signal. No expression was detected by in situ RT-PCR in two Influenza-like Illness A (H1N1)-negative patients using standard protocols nor in a non-related cervical cell line. In situ relative transcription levels correlated with those obtained when in vitro RT-PCR assays were performed. Partial sequences of the NA gene from A (H1N1)-positive patients were obtained by the in situ RT-PCR-sequencing method. Sequence analysis showed 98% similarity with influenza viruses reported previously in other places. CONCLUSIONS: We have successfully amplified specific influenza A (H1N1) NA sequences using stored clinical material; results suggest that this strategy could be useful when clinical RNA samples are quantity limited, or when poor quality is obtained. Here, we provide a very sensitive method that specifically detects the neuraminidase viral RNA in lung samples from patients who died from pneumonia caused by Influenza A (H1N1) outbreak in Mexico City.