The Role of miRNAs to Detect Progression, Stratify, and Predict Relevant Clinical Outcomes in Bladder Cancer.

Bladder cancer (BC) is one of the most common types of cancer worldwide, with significant differences in survival depending on the degree of muscle and surrounding tissue invasion. For this reason, the timely detection and monitoring of the disease are important. Surveillance cystoscopy is an invasive, costly, and uncomfortable procedure to monitor BC, raising the need for new, less invasive alternatives. In this scenario, microRNAs (miRNAs) represent attractive prognostic tools given their role as gene regulators in different biological processes, tissue expression, and their ease of evaluation in liquid samples. In cancer, miRNA expression is dynamically modified depending on the tumor type and cancer staging, making them potential biomarkers. This review describes the most recent studies in the last five years exploring the utility of miRNA-based strategies to monitor progression, stratify, and predict relevant clinical outcomes of bladder cancer. Several studies have shown that multimarker miRNA models can better predict overall survival, recurrence, and progression in BC patients than traditional strategies, especially when combining miRNA expression with clinicopathological variables. Future studies should focus on validating their use in different cohorts and liquid samples.

Expression of ornithine decarboxylase in peripheral blood mononuclear cells from patients with pancreatic adenocarcinoma: A preliminary report.

Ductal adenocarcinoma represents 90-95% of pancreatic cancer (PC) cases and it is an aggressive disease with asymptomatic evolution at early stages, non-specific symptoms and a typical late diagnosis with a 5-year survival rate estimated to be 8%. A window of opportunity lies in early diagnosis as there are currently no reliable biomarkers. CA 19-9 is one of the most frequently used biomarkers of PC, with 75 and 77.6% sensitivity (Se) and specificity (Sp), respectively, and the carcinoembryonic antigen (CEA) shows 39.5 and 81.3% of Se and Sp, respectively. A case-control study was conducted including adult patients with a histological diagnosis of PC (n=11) without previous treatment at the Oncology Service of the CMNO-IMSS between 2019 and 2020, and a control group of adult volunteers (n=11) who were clinically healthy or with controlled disease including hypertension, hypothyroidism and diabetes. Clinical, laboratory and sociodemographic data as well as blood, urine and saliva samples were collected following patient consent. Polyamines were quantified using high-performance liquid chromatography with fluorescence detection, CA 19-9 and CEA were evaluated using enzyme-linked immunosorbent assay, and the protein expression of ornithine decarboxylase (ODC) was evaluated using western blotting. Polyamine metabolism and modulation by means of ODC were increased in the serum and saliva of patients with PC, and the expression of ODC alone was increased in peripheral blood mononuclear cells (PBMCs). The present study focused on the evaluation of putrescine, spermine, spermidine and ODC in PBMCs associated with CA 19-9 and CEA as an auxiliary tool in PC diagnosis.

HPV-Negative and HPV-Positive Oral Cancer Cells Stimulate the Polarization of Neutrophils towards Different Functional Phenotypes In Vitro.

High-risk human papillomavirus (HPV) infection is one of the leading causes of oropharyngeal squamous cell carcinoma (OPSCC), while the correlation between HPV and oral squamous cell carcinoma (OSCC) remains controversial. The inflammatory infiltrate involved in these epithelial neoplasms differs based on their association with HPV. HPV- tumors show higher tumor-associated neutrophil (TAN) infiltration. It is believed that TANs can play a dual role in cancer by exerting either anti-tumorigenic or pro-tumorigenic effects. However, the impact of HPV status on neutrophil polarization remains unknown. Therefore, this study aimed to investigate the effect of OSCC cells, both HPV- and HPV16+, on the functional phenotype of neutrophils. Peripheral blood neutrophils were stimulated with supernatants from OSCC cell lines and non-tumorigenic HaCaT keratinocytes transduced with HPV16 E6/E7 oncogenes. Subsequently, cytokine production, cell viability, metabolism, expression of degranulation markers, and PD-L1 expression were evaluated. Our findings demonstrate that in contrast to UPCI:SCC154 (HPV+ OSCC) cells, the SCC-9 (HPV- OSCC) cell line induced a highly activated functional state in neutrophils, which is potentially associated with a pro-tumorigenic effect. The HaCaT 16-E7 supernatant only stimulated the activation of some neutrophil functions. Understanding the complex interplay between neutrophils and their microenvironment has the potential to identify TANs as viable therapeutic targets.

BAFF system expression in double negative 2, activated naïve and activated memory B cells in systemic lupus erythematosus.

Introduction: B cell activating factor (BAFF) has an important role in normal B cell development. The aberrant expression of BAFF is related with the autoimmune diseases development like Systemic Lupus Erythematosus (SLE) for promoting self-reactive B cells survival. BAFF functions are exerted through its receptors BAFF-R (BR3), transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA) that are reported to have differential expression on B cells in SLE. Recently, atypical B cells that express CD11c have been associated with SLE because they are prone to develop into antibody-secreting cells, however the relationship with BAFF remains unclear. This study aims to analyze the BAFF system expression on CXCR5- CD11c+ atypical B cell subsets double negative 2 (DN2), activated naïve (aNAV), switched memory (SWM) and unswitched memory (USM) B cells. Methods: Forty-five SLE patients and 15 healthy subjects (HS) were included. Flow cytometry was used to evaluate the expression of the receptors in the B cell subpopulations. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify the soluble levels of BAFF (sBAFF) and IL-21. Results: We found increased frequency of CXCR5- CD11c+ atypical B cell subpopulations DN2, aNAV, SWM and USM B cells in SLE patients compared to HS. SLE patients had increased expression of membrane BAFF (mBAFF) and BCMA receptor in classic B cell subsets (DN, NAV, SWM and USM). Also, the CXCR5+ CD11c- DN1, resting naïve (rNAV), SWM and USM B cell subsets showed higher mBAFF expression in SLE. CXCR5- CD11c+ atypical B cell subpopulations DN2, SWM and USM B cells showed strong correlations with the expression of BAFF receptors. The atypical B cells DN2 in SLE showed significant decreased expression of TACI, which correlated with higher IL-21 levels. Also, lower expression of TACI in atypical B cell DN2 was associated with high disease activity. Discussion: These results suggest a participation of the BAFF system in CXCR5- CD11c+ atypical B cell subsets in SLE patients. Decreased TACI expression on atypical B cells DN2 correlated with high disease activity in SLE patients supporting the immunoregulatory role of TACI in autoimmunity.

Pentoxifylline Inhibits TNF-α/TGF-ß1-Induced Epithelial-Mesenchymal Transition via Suppressing the NF-κB Pathway and SERPINE1 Expression in CaSki Cells.

Cervical cancer (CC) is one of the most common and deadly types of female cancer worldwide. Late diagnosis in CC increases the risk of tumor cells spreading to distant organs (metastasis). The epithelial-mesenchymal transition (EMT) is a fundamental process of cancer metastasis. Inflammation can lead to tumor progression, EMT induction, and metastasis. The inflammatory microenvironment is a potent inducer of EMT; inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-α) and Transforming growth factor-beta (TGF-ß1) activate transcriptional factors such as STAT3, Snail, Smad, and the Nuclear Factor kappa light-chain-enhancer of activated beta cells (NF-κΒ), which drive EMT. Anti-inflammatory compounds may be an option in the disruption of EMT. PenToXifylline (PTX) possesses potent anti-inflammatory effects by inhibiting NF-κB activity. In addition, PTX exerts an anti-fibrotic effect by decreasing Smad2/3/4. We hypothesize that PTX could exert anti-EMT effects. CaSki human cervical tumor cells were exposed to TNF-α 10 ng/mL and TGF-ß1 alone or in combination for 5 days. Our results revealed that TNF-α and TGF-ß1 induced N-cadherin and Vimentin, confirming the induction of EMT. Furthermore, the combination of cytokines synergized the expression of mesenchymal proteins, enhanced IκBα and p65 phosphorylation, and upregulated Serpin family E member 1 (SERPINE1) mRNA. PTX pretreatment prior to the addition of TNF-α and TGF-ß1 significantly reduced N-cadherin and Vimentin levels. To our knowledge, this is the first time that this effect of PTX has been reported. Additionally, PTX reduced the phosphorylation of IκB-α and p65 and significantly decreased SERPINE1 expression, cell proliferation, migration, and invasion. In conclusion, PTX may counteract EMT in cervical cancer cells by decreasing the NF-κB and SERPINE1.

A Modified Method for the Quantification of Immune Checkpoint Ligands on Exosomes from Human Serum using Flow Cytometry.

Objectives: Exosomes are the smallest of the extracellular vesicles and can contain a variety of different cargos, including nucleic acids, lipids, and proteins. Ultracentrifugation followed by electron microscopy has historically been used for the isolation and visualization of exosomes; Western blot and ELISA have also been used, but these techniques are only semiquantitative and are unable to distinguish different exosome markers in the same sample. To resolve some of these issues, we propose a modification of a bead-based flow cytometry method. Methods: Peripheral blood serum was mixed with a commercial exosome separation reagent and incubated for 30 min at 4°, centrifuged, exosome pellet was isolated and resuspended in PBS. Exosomes were then added to magnetic beads, incubated 18 h, then incubated with exosome-specific antibodies for 1 h. The resulting bead:exosome complexes were centrifuged and then washed, then washed again using a magnetic separator, resuspended in PBS, and analyzed via flow cytometry. Results: Using commercial magnetic beads bound with anti-CD63, our protocol modifies starting conditions, washing steps, and magnetic separation and uses the FSC and SSC determination of the flow cytometer to result in increased yield and identification of the exosome populations of interest. Our modified protocol increased the yield of specific populations approximately 10-fold. Conclusion: The new protocol was used to identify exosomes positive for 2 immune checkpoint ligands in serum-derived exosomes from cervical cancer patients. We suspect that this protocol can also be used for the identification of other exosome proteins since we also quantified the exosome membrane-enriched tetraspanins CD9 and CD81. Identification of proteins rarely expressed in exosomes is complicated in this technique as serum is an inherently dirty source of exosomes, and great care must be taken in the washing and gating of the exosome:bead populations.

The role of miRNA in prostate cancer diagnosis, prognosis and treatment response: a narrative review.

To date, prostate cancer remains the most common tumor diagnosed in males and the second most common cause of cancer-related mortality. While current screening protocols can detect early disease, they lack enough sensitivity and specificity, leading to unnecessary biopsies and overtreatment. Furthermore, disease monitoring remains challenging, as current prognostic strategies rely on data obtained by invasive means such as biopsy, surgery and digital rectal examination. Additionally, there are no tools to predict tumor progression, risk reclassification and treatment response. As the need for accurate biomarkers continues, miRNAs are promising biomarkers for screening, surveillance, prognosis and treatment response in prostate cancer. In this review, the authors describe the current evidence regarding the accuracy and efficacy of these biomarkers for prostate cancer.

Comparative Genomic Hybridization and Transcriptome Sequencing Reveal Genes with Gain in Acute Lymphoblastic Leukemia: JUP Expression Emerges as a Survival-Related Gene.

Acute lymphoblastic leukemia (ALL) in children or adults is characterized by structural and numeric aberrations in chromosomes; these anomalies strongly correlate with prognosis and clinical outcome. Therefore, this work aimed to identify the genes present in chromosomal gain regions found more frequently in patients with acute lymphoblastic leukemia (ALL) and ALL-derived cell lines using comparative genomic hybridization (CGH). In addition, validation of the genes found in these regions was performed utilizing RNAseq from JURKAT, CEM, and SUP-B15 cell lines, as well as expression microarrays derived from a MILE study. Chromosomes with common gain zones that were maintained in six or more samples were 14, 17, and 22, in which a total of 22 genes were identified. From them, NT5C3B, CNP, ACLY, and GNB1L maintained overexpression at the mRNA level in the cell lines and in patients with ALL. It is noteworthy that SALL2 showed very high expression in T-ALL, while JUP was highly expressed in B-ALL lineages. Interestingly, the latter correlated with worse survival in patients. This provided evidence that the measurement of these genes has high potential for clinical utility; however, their expressions should first be evaluated with a sensitive test in a more significant number of patients.

Human lactoferrin from breast milk: characterization by HPLC and its in vitro antibiofilm performance.

Preterm infants are at high risk of infection due to opportunistic bacteria as Pseudomonas aeruginosa, causing infections among infants in neonatal intensive care units. Human lactoferrin (hLf) is a multifunctional protein and one of the most abundant in breast milk, and plays an important role in prevention of different infections in neonates. This work offers a strategy to obtain a lyophilisate of purified lactoferrin from breast milk. In addition, a reliable HPLC method for quantification of lactoferrin with a linear quantification range of 0.040-0.140 mg/mL with selectivity, accuracy and repeatability, is described. Lyophilized hLf was obtained by purification through a heparin affinity column followed by ultrafiltration with a 30 kDa membrane. The final solution was lyophilized and the product was analyzed using HPLC method, recovering about 70% of initial lactoferrin in the sample. This molecule was elucidated through FTIR spectroscopy and SDS-PAGE electrophoresis. In addition, the capacity against biofilm formation of P. aeruginosa was demonstrated with 75% of inhibition at 6 mg/mL. These results suggest that lyophilized hLf can be obtained by purification of breast milk and that it can provide antibiofilm activity against P. aeruginosa.

Loss of mitochondrial membrane potential (ΔΨm ) in leucocytes as post-COVID-19 sequelae.

The mitochondrial membrane potential (ΔΨm ) is a parameter often used to determine mitochondrial function; therefore, it can be used to determine the integrity and functionality of cells. A decrement of ΔΨm is implicated in several inflammatory-related pathologies, such phenomena can be related to COVID-19 infection. The present work aimed to compare the ΔΨm in leucocytes (human PBMCs; HPBMC) isolated from healthy control (HC) subjects, patients with COVID-19 (C-19), recovered subjects at 40 ± 13 (R1) and 335 ± 20 (R2) days after infection (dai). Obtained data showed that ΔΨm decreased in HPBMC of subjects with C-19, R1, and R2 compared with HC. When analyzing the ΔΨm data by sex, in females, a significant decrease was observed in R1 and R2 groups versus HC. Regarding men, a significant decrease of ΔΨm was observed in R1, with respect to HC, contrary to R2 group, who reestablished this parameter. Obtained results suggest that the loss of ΔΨm could be related to the long-COVID.

Sensitizing the cytotoxic action of Docetaxel induced by Pentoxifylline in a PC3 prostate cancer cell line.

BACKGROUND: Prostate cancer is one of the most frequently diagnosed types of cancers worldwide. In its initial period, the tumor is hormone-sensitive, but in advanced states, it evolves into a metastatic castration-resistant tumor. In this state, chemotherapy with taxanes such as Docetaxel (DTX) comprises the first line of treatment. However, the response is poor due to chemoresistance and toxicity. On the other hand, Pentoxifylline (PTX) is an unspecific inhibitor of phosphodiesterases; experimental, and clinically it has been described as sensitizing tumor cells to chemotherapy, increasing apoptosis and decreasing senescence. We study whether the PTX sensitizes prostate cancer cells to DTX for greater effectiveness. METHODS: PC3 human prostate cancer cells were treated in vitro at different doses and times with PTX, DTX, or their combination. Viability was determined by the WST-1 assay by spectrophotometry, cell cycle progression, apoptosis, generic caspase activation and senescence by flow cytometry, DNA fragmentation and caspases-3, -8, and -9 activity by ELISA. RESULTS: We found that PTX in PC3 human prostate cancer cells induces significant apoptosis per se and increases that generated by DTX, while at the same time it reduces the senescence caused by the chemotherapy and increases caspases-3,-8, and -9 activity in PTX + DTX-treated cells. Both treatments blocked the PC3 cell in the G1 phase. CONCLUSIONS: Our results show that PTX sensitizes prostate tumor cells to apoptosis induced by DTX. Taken together, the results support the concept of chemotherapy with rational molecular bases.

B7-H6, an immunoligand for the natural killer cell activating receptor NKp30, reveals inhibitory effects on cell proliferation and migration, but not apoptosis, in cervical cancer derived-cell lines.

BACKGROUND: Although great progress has been made in treatment regimens, cervical cancer remains as one of the most common cancer in women worldwide. Studies focusing on molecules that regulate carcinogenesis may provide potential therapeutic strategies for cervical cancer. B7-H6, an activating immunoligand expressed by several tumor cells, is known to activate NK cell-mediated cytotoxicity once engaged with its natural receptor NKp30. However, the opposite, that is, the effects in the tumor cell triggered by B7-H6 after interacting with NKp30 has not yet been well explored. METHODS: In this study, we evaluated the surface expression of B7-H6 by flow cytometry. Later, we stimulated B7-H6 positive cervical cancer derived-cell lines (HeLa and SiHa) with recombinant soluble NKp30 (sNKp30) protein and evaluated biological effects using the impedance RTCA system for cell proliferation, the scratch method for cell migration, and flow cytometry for apoptosis. Cellular localization of B7-H6 was determined using confocal microscopy. RESULTS: Notably, we observed that the addition of sNKp30 to the cervical cancer cell lines decreased tumor cell proliferation and migration rate, but had no effect on apoptosis. We also found that B7-H6 is selectively maintained in tumor cell lines, and that efforts to sort and purify B7-H6 negative or positive cells were futile, as negative cells, when cultured, regained the expression of B7-H6 and B7-H6 positive cells, when sorted and cultivated, lost a percentage of B7-H6 expression. CONCLUSIONS: Our results suggest that B7-H6 has an important, as of yet undescribed, role in the biology of the cervical tumor cells themselves, suggesting that this protein might be a promising target for anti-tumor therapy in the future.

Differential effects of alliin and allicin on apoptosis and senescence in luminal A and triple-negative breast cancer: Caspase, ΔΨm, and pro-apoptotic gene involvement.

Breast cancer is the most frequent cancer in women worldwide, and drug resistance is common in all breast cancer types. The combination of natural products with chemotherapies has attracted attention, as it was found that natural compounds enhance the effects of standard cancer chemotherapeutic drugs and protect from side effects. Into the different natural products, garlic has been recognized for its antitumor properties. It is suggested that its anticancer effects are associated with its organo-sulfur compounds, especially alliin and allicin. Here, we evaluated the effects of both molecules on cell death, senescence, and their senolytic potential in luminal A and triple-negative breast cancer cells. MCF-7 (luminal A) and HCC-70 (triple-negative) cells were cultured and treated with different concentrations of alliin or allicin. Then, cell viability was determined using the WST-1 reagent. Apoptosis and caspase activity were evaluated by flow cytometry; ΔΨm was assessed using a JC-10 fluorometric assay kit. Apoptosis-related genes were evaluated by RT-PCR. Proliferation was measured using bromodeoxyuridine incorporation. We also evaluated clonogenicity, senescence (ß-Galactosidase Staining), and the senolytic effect of the compounds. Our results showed that allicin has antiproliferative, anticlonogenic, and senolytic effects. In addition, allicin decreased cell viability and induced apoptosis by loss of ΔΨm, caspase-3, caspase-8, and caspase-9 activation, upregulation of NOXA, P21, and BAK, as well as downregulation of BCL-XL expression. Contrary to allicin, alliin promoted clonogenicity, induced senescence, and did not exhibit pro-apoptotic effects in breast cancer cells.

Pentoxifylline Sensitizes Cisplatin-Resistant Human Cervical Cancer Cells to Cisplatin Treatment: Involvement of Mitochondrial and NF-Kappa B Pathways.

BACKGROUND: Cervical cancer continues to be a major public health problem worldwide, and Cisplatin is used as first-line chemotherapy for this cancer; however, malignant cells exposed to CISplatin (CIS) become insensitive to the effects of this drug. PenToXifylline (PTX) is a xanthine that sensitizes several types of tumor cells to apoptosis induced by antitumor drugs, such as Adriamycin, Carboplatin, and CIS. The effects of PTX on tumor cells have been related to the disruption of the NF-κB pathway, thus preventing the activation of cell survival mechanisms such as the expression of anti-apoptotic genes, the secretion of proinflammatory interleukins, and growth factors. OBJECTIVE: In this work, we studied the antitumor proprieties of PTX in human SiHa cervical carcinoma cells resistant to CIS. MATERIALS AND METHODS: SiHa and HeLa cervical cancer cells and their CIS-resistant derived cell lines (SiHaCIS-R and HeLaCIS-R, respectively) were used as in-vitro models. We studied the effects of PTX alone or in combination with CIS on cell viability, apoptosis, caspase-3, caspase-8, and caspase-9 activity, cleaved PARP-1, anti-apoptotic protein (Bcl-2 and Bcl-xL) levels, p65 phosphorylation, cadmium chloride (CdCl2) sensitivity, Platinum (Pt) accumulation, and glutathione (GSH) levels, as well as on the gene expression of GSH and drug transporters (influx and efflux). RESULTS: PTX sensitized SiHaCIS-R cells to the effects of CIS by inducing apoptosis, caspase activation, and PARP-1 cleavage. PTX treatment also decreased p65 phosphorylation, increased Pt levels, depleted GSH, and downregulated the expression of the ATP7A, ATP7B, GSR, and MGST1 genes. CONCLUSION: PTX reverses the acquired phenotype of CIS resistance close to the sensitivity of parental SiHa cells.

17ß­estradiol­induced mitochondrial dysfunction and Warburg effect in cervical cancer cells allow cell survival under metabolic stress.

Mitochondria from different types of cancer show bioenergetics and dysfunction that favor cell proliferation. The mechanistic understanding of estrogen in cervical cancer is poorly understood. Therefore, the objective of this study was to determine how 17ß­estradiol (E2) affects mitochondrial function and the Warburg effect in SiHa, HeLa and C33A cervical cancer cells. Mitochondrial compromise was evaluated measuring changes in the membrane permeability by immunofluorescence, calcium concentration, redox status, iron and ferritin reserves. Glucose consumption and lactic acid assays were used to detect the metabolic activity. Results were confirmed at molecular level by analysis of the differential gene expression using RNA sequencing. E2 modified the mitochondrial permeability and produced an alteration in the calcium signaling pathway. In HeLa and SiHa, there was a significant decrease in nitric oxide levels and lipid peroxidation, and an increase in glucose consumption and lactic acid levels when stimulated with E2. Intracellular iron or ferritin reserves were not affected by the E2 treatment. Genes differentially modulated by E2 were involved in the mitochondrial electron transport chain, oxidative phosphorylation system, glycolysis, pentose phosphate pathway and the regulation of metabolic signaling pathways. Herein, we provide evidence for a primary effect of estrogen on mitochondrial function and the Warburg effect, favoring the metabolic adaptation of the cervical cancer cell lines and their survival.

Low-dose endosulfan inhibits proliferation and induces senescence and pro-inflammatory cytokine production in human lymphocytes, preferentially impacting cytotoxic cells.

Endosulfan is a DDT-era organochlorine pesticide. Due to past and current environmental contamination, investigation of endosulfan exposure is of current importance. Acute high dose exposure precipitates neural/endocrine system damage, but the effects on the immune system and of lower doses are not well-characterized. Two relatively low concentrations of endosulfan (i.e. 0.1 and 17 µM ENDO) were investigated in an in vitro study using human peripheral blood mononuclear cells (PBMC) to understand effects of relatively low doses (0.1-25.0 µM [≈0.04-10 ppm/40-10,000 ppb]) of ENDO upon normal human T- and B-lymphocytes and NK cells. The study here found that 17 µM ENDO inhibited phytohemagglutinin-M (PHA)-induced human PBMC proliferation. It was also seen that senescence and apoptosis among non-stimulated cells was increased, specifically within CD8 and NK populations, and that CD4:CD8 ratios also were increased. Treatment of non-stimulated PBMC with ENDO led to overall increases in production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, -4, and -6, and decreased production of anti-inflammatory IL-10, suggesting an immunosenescence secretory phenotype. Interestingly, when the cells were pre-stimulated with mitogen (PHA), ENDO became inhibitory against the mitogen-induced proliferation and cytokine formation - with the exception of that of TNFα and IL-6, suggesting differential effects of ENDO on activated cells. Thus, at the organismal level, ENDO might also display differential effects during states of autoimmune disease or chronic viral infection in the exposed host.

Pentoxifylline Enhances the Apoptotic Effect of Carboplatin in Y79 Retinoblastoma Cells.

BACKGROUND/AIM: Retinoblastoma (RB) is the most common primary intraocular malignancy. Carboplatin (CPt) is a DNA damage-inducing agent that is widely used for the treatment of RB. Unfortunately, this drug also activates the transcription factor nuclear factor-kappa B (NF-ĸB), leading to promotion of tumor survival. Pentoxifylline (PTX) is a drug that inhibits the phosphorylation of I kappa B-alpha (IĸBα) in serines 32 and 36, and this disrupts NF-ĸB activity that promotes tumor survival. The goal of this study was to evaluate the effect of the PTX on the antitumor activity of CPt. MATERIALS AND METHODS: Y79 RB cells were treated with CPt, PTX, or both. Cell viability, apoptosis, loss of mitochondrial membrane potential, the activity of caspase-9, -8, and -3, cytochrome c release, cell-cycle progression, p53, and phosphorylation of IĸBα, and pro- and anti-apoptotic genes were evaluated. RESULTS: Both drugs significantly affected the viability of the Y79 RB cells in a time- and dose-dependent manner. The PTX+CPt combination exhibited the highest rate of apoptosis, a decrease in cell viability and significant caspase activation, as well as loss of mitochondrial membrane potential, release of cytochrome c, and increased p53 protein levels. Cells treated with PTX alone displayed decreased I kappa B-alpha phosphorylation, compared to the CPt treated group. In addition, the PTX+CPt combination treatment induced up-regulation of the proapoptotic genes Bax, Bad, Bak, and caspases- 3, -8, and -9, compared to the CPt and PTX individual treated groups. CONCLUSION: PTX induces apoptosis per se and increases the CPt-induced apoptosis, augmenting its antitumor effectiveness.

Effects of 60 kDa prolactin and estradiol on metabolism and cell survival in cervical cancer: Co­expression of their hormonal receptors during cancer progression.

Estrogens and estrogen receptors (ERs), such as ERα and ERß, prolactin (PRL) and prolactin receptor (PRLR) have been reported to be involved in the physiopathology of uterine cervical cancer (UCC). The 60 kDa PRL is an isoform of PRL, which is produced by UCC­derived cells. The present study aimed to evaluate the expression of hormonal receptors in different degrees of cervical lesions, and to determine whether 60 kDa PRL and 17ß­estradiol (E2) modulated cell survival and metabolism in UCC cells, and in HaCaT cells transduced with human papillomavirus (HPV) 16 and 18 E6/E7 oncogenes. ERα, ERß, PRLR, Ki67 and B­cell lymphoma 2 expression levels were analyzed in biopsies of precursor lesions and UCC using immunohistochemistry. In addition, HeLa, SiHa and C33A cells, and transduced HaCaT cells, were stimulated with 60 kDa PRL, E2 or a combination of both. Proliferation was evaluated using the xCELLigence platform, apoptosis was analyzed by flow cytometry and cell metabolism was determined using the MTT assay. The results revealed that ERα, ERß, PRLR and Ki67 expression levels were increased during the progression of cancer. In vitro, 60 kDa PRL alone significantly increased proliferation of SiHa cells. Furthermore, E2 alone or in combination with 60 kDa PRL increased the sensitivity of SiHa cells to cisplatin and increased the percentage of apoptosis; in HaCaT cells, these treatment strategies had the opposite effect on cisplatin sensitivity. Treatment with E2 increased mitochondrial activity in HeLa and SiHa cells, and in HaCaT cells transduced with HPV 16 E6/E7 and HPV 18 E6 oncogenes. PRL had a similar effect on HeLa cells, and on HaCaT cells transduced with HPV 18 E6 and HPV 16 E7. The co­expression of these receptors demonstrated the hormonal dependence of UCC. In addition, E2 and the 60 kDa PRL significantly impacted the metabolism, but not the survival, of cells.

Urine TREM-1 as a marker of urinary tract infection in children.

Objective Triggering receptor expressed on myeloid cells (TREM)-1 is a receptor that is thought to improve recognition of patients with true infection. In this study, we investigated whether Triggering receptor expressed on myeloid cells (TREM-1) is present in urine samples from children with urinary tract infection (UTI) and in samples from healthy children. Methods A total of 128 samples met the inclusion criteria for the study. Urine samples were processed for culture and urinalysis as a regular protocol for patients with UTI. Samples were classified according to culture and urinalysis results. TREM-1 protein expression was detected with flow cytometry and sTREM-1 was assessed by ELISA. Results Flow cytometry showed detectable expression of TREM-1 in 100% of samples, UTI and non-UTI groups ( p < 0.001). Mean fluorescence intensity of TREM-1 was different between the groups ( p < 0.001). Levels of sTREM-1 were detected in patients with UTI, but not in non-UTI patients. Conclusions All of our patients (healthy and diseased) showed TREM-1 expression. However, TREM-1 levels in patients with UTI tend to be higher and are associated with increased neutrophils and cytokine activity induced by bacteria.