Susana del Toro-Arreola

Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions

BackgroundPersistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection.MethodsNKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays.ResultsWe report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients.ConclusionOur results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.

Augmented serum level of major histocompatibility complex class I-related chain A (MICA) protein and reduced NKG2D expression on NK and T cells in patients with cervical cancer and precursor lesions

BackgroundCervical cancer is the second most common cancer in women worldwide. NK and cytotoxic T cells play an important role in the elimination of virus-infected and tumor cells through NKG2D activating receptors, which can promote the lysis of target cells by binding to the major histocompatibility complex class I-related chain A (MICA) proteins. Increased serum levels of MICA have been found in patients with epithelial tumors. The aim of this study was to compare the levels of soluble MICA (sMICA) and NKG2D-expressing NK and T cells in blood samples from patients with cervical cancer or precursor lesions with those from healthy donors.MethodsPeripheral blood with or without heparin was collected to obtain mononuclear cells or sera, respectively. Serum sMICA levels were measured by ELISA and NKG2D-expressing immune cells were analyzed by flow cytometry. Also, a correlation analysis was performed to associate sMICA levels with either NKG2D expression or with the stage of the lesion.ResultsSignificant amounts of sMICA were detected in sera from nearly all patients. We found a decrease in the number of NKG2D-expressing NK and T cells in both cervical cancer and lesion groups when compared to healthy donors. Pearson analysis showed a negative correlation between sMICA and NKG2D-expressing T cells; however, we did not find a significant correlation when the analysis was applied to sMICA and NKG2D expression on NK cells.ConclusionOur results show for the first time that high sMICA levels are found in sera from patients with both cervical cancer and precursor lesions when compared with healthy donors. We also observed a diminution in the number of NKG2D-expressing NK and T cells in the patient samples; however, a significant negative correlation between sMICA and NKG2D expression was only seen in T cells.

Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence

BackgroundWorldwide, cervical cancer is the second most common causes of cancer in women and represents an important mortality rate. Cisplatin (CIS) is a very important antitumoral agent and can lead tumor cells toward two important cellular states: apoptosis and senescence. In some types of cancers pentoxifylline (PTX) sensitizes these cells to the toxic action of chemotherapeutics drugs such as adriamycin, inducing apoptosis. In the present work, we studied in vitro whether PTX alone or in combination with CIS induces apoptosis and/or senescence in cervix cancer HeLa and SiHa cell lines infected with HPV types 16 and 18, respectively, as well as in immortalized keratinocytyes HaCaT cells.MethodsHeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, CIS or both. The cellular toxicity and survival fraction of PTX and CIS were determinate by WST-1 and clonogenic assays respectively. Apoptosis, caspase activation and phosphorylation of ERK1/2, p38, p65 (NF-κB), Bcl-2 and Bcl-XL anti-apoptotic proteins were determinated by flow cytometry. Senescence by microscopy. Phosphorylation of IκBα and IκB total were measured by ELISA. Pro-apoptotic, anti-apoptotic and senescence genes, as well as HPV-E6/7 mRNA expression, were detected by RT-PCR.ResultsOur results show that after 24 hours of incubation PTX per se is toxic for cancer cells affecting cell viability and inducing apoptosis. The toxicity in HaCaT cells was minimal. CIS induces apoptosis in HeLa and SiHa cells and its effect was significantly increases when the cells were treated with PTX + CIS. In all studies there was a direct correlation with levels of caspases (-3, -6, -7, -9 and -8) activity and apoptosis. CIS induces important levels of senescence and phosphorylation of ERK1/2, p38, p65/RELA, and IκBα, and decreased the expression of anti-apoptotic protein Bcl-XL. Surprisingly these levels were significantly reduced by PTX in tumor cells, and at the same time, increases the expression of pro-apoptotic genes.ConclusionPTX sensitizes cervical cancer cells to CIS-induced apoptosis and decreases the CIS-induced senescence in these cells via inhibition of NF-κB signaling pathway; diminishes expression of antiapoptotic proteins and the activation of caspases.

Sensitization of cervix cancer cells to Adriamycin by Pentoxifylline induces an increase in apoptosis and decrease senescence

BackgroundChemotherapeutic drugs like Adriamycin (ADR) induces apoptosis or senescence in cancer cells but these cells often develop resistance and generate responses of short duration or complete failure. The methylxantine drug Pentoxifylline (PTX) used routinely in the clinics setting for circulatory diseases has been recently described to have antitumor properties. We evaluated whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cells.MethodsHeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, ADR or PTX + ADR. The cellular toxicity of PTX and survival fraction were determinated by WST-1 and clonogenic assay respectively. Apoptosis, caspase activation and ADR efflux rate were measured by flow cytometry, senescence by microscopy. IκBα and DNA fragmentation were determinated by ELISA. Proapoptotic, antiapoptotic and senescence genes, as well as HPV-E6/E7 mRNA expression, were detected by time real RT-PCR. p53 protein levels were assayed by Western blot.ResultsPTX is toxic (WST-1), affects survival (clonogenic assay) and induces apoptosis in cervix cancer cells. Additionally, the combination of this drug with ADR diminished the survival fraction and significantly increased apoptosis of HeLa and SiHa cervix cancer cells. Treatments were less effective in HaCaT cells. We found caspase participation in the induction of apoptosis by PTX, ADR or its combination. Surprisingly, in spite of the antitumor activity displayed by PTX, our results indicate that methylxantine, per se does not induce senescence; however it inhibits senescence induced by ADR and at the same time increases apoptosis. PTX elevates IκBα levels. Such sensitization is achieved through the up-regulation of proapoptotic factors such as caspase and bcl family gene expression. PTX and PTX + ADR also decrease E6 and E7 expression in SiHa cells, but not in HeLa cells. p53 was detected only in SiHa cells treated with ADR.ConclusionPTX is a good inducer of apoptosis but does not induce senescence. Furthermore, PTX reduced the ADR-induced senescence and increased apoptosis in cervix cancer cells.

PROCALCITONIN AND C-REACTIVE PROTEIN SERUM LEVELS AS MARKERS OF INFECTION IN A PEDIATRIC POPULATION WITH FEBRILE NEUTROPENIA AND CANCER

Background: Procalcitonin and C-reactive-protein are inflammatory markers for sepsis. The authors evaluated their sensitivity and specificity in pediatric patients with cancer and febrile neutropenia. Procedure: Serum procalcitonin and C-reactive-protein were evaluated. Patients (n = 54) were divided into 2 groups, with severe infection (n = 18) or without documented infection (n = 36). Results: Procalcitonin and C-reactive protein were significantly higher in the high-risk group. Procalcitonin displayed 72.2% sensitivity and 80.5% specificity. C-reactive-protein had a sensitivity of 77.7% and specificity of 77.2%. Conclusions: Procalcitonin is an accurate predictor of bacterial infection in neutropenic children, while C-reactive-protein may be a better screening test in emergency settings.

Lipopolysaccharide induces the expression of an autocrine prolactin loop enhancing inflammatory response in monocytes

BackgroundProlactin from pituitary gland helps maintain homeostasis but it is also released in immune cells where its function is not completely understood. Pleiotropic functions of prolactin (PRL) might be mediated by different isoforms of its receptor (PRLr).MethodsThe aim of this study was to investigate the relationship between the eventual synthesis of PRL and PRLr isoforms with the inflammatory response in monocytes. We used THP-1 and monocytes isolated from healthy subjects stimulated with lipopolysaccharide (LPS). Western blot, real time PCR and immunocytochemistry were performed to identify both molecules. The bioactivity of the PRL was assessed using a bioassay and ELISA to detect pro inflammatory cytokines.ResultsPRLr mRNA and PRL mRNA were synthesized in THP-1 monocytes activated with LPS with peaks of 300-fold and 130-fold, respectively. The long (100 kDa) and the intermediate (50 kDa) isoforms of PRLr and big PRL (60 kDa) were time-dependent upregulated for monocytes stimulated with LPS. This expression was confirmed in monocytes from healthy subjects. The PRLr intermediate isoform and the big PRL were found soluble in the culture media and later in the nucleus in THP-1 monocytes stimulated with LPS. Big PRL released by monocytes showed bioactivity in Nb2 Cells, and both PRL and PRLr, synthesized by monocytes were related with levels of nitrites and proinflammatory citokines.ConclusionsOur results suggest the expression of a full-autocrine loop of PRL enhances the inflammatory response in activated monocytes. This response mediated by big PRL may contribute to the eradication of potential pathogens during innate immune response in monocytes but may also contribute to inflammatory disorders.

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

BackgroundCervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity.ResultsWe demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells.ConclusionsOur results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

MHC class I-related chain A and B ligands are differentially expressed in human cervical cancer cell lines

BackgroundNatural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line.ResultsHere we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress.ConclusionsThis study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells.

High expression of prolactin receptor is associated with cell survival in cervical cancer cells

BackgroundThe altered expression of prolactin (PRL) and its receptor (PRLR) has been implicated in breast and other types of cancer. There are few studies that have focused on the analysis of PRL/PRLR in cervical cancer where the development of neoplastic lesions is influenced by the variation of the hormonal status. The aim of this study was to evaluate the expression of PRL/PRLR and the effect of PRL treatment on cell proliferation and apoptosis in cervical cancer cell lines.ResultsHigh expression of multiple PRLR forms and PRLvariants of 60–80 kDa were observed in cervical cancer cell lines compared with non-tumorigenic keratinocytes evaluated by Western blot, immunofluorecence and real time PCR. Treatment with PRL (200 ng/ml) increased cell proliferation in HeLa cells determined by the MTT assay at day 3 and after 1 day a protective effect against etoposide induced apoptosis in HeLa, SiHa and C-33A cervical cancer cell lines analyzed by the TUNEL assay.ConclusionsOur data suggests that PRL/PRLR signaling could act as an important survival factor for cervical cancer. The use of an effective PRL antagonist may provide a better therapeutic intervention in cervical cancer.

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

BackgroundThe NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured.ResultsClose to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group.ConclusionsTaken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.