Metformin in combination with chemotherapy increases apoptosis in gastric cancer cells and counteracts senescence induced by chemotherapy.

Gastric cancer (GC) is the fourth leading cause of cancer death in the world, and there is a demand for new therapeutic agents to treat GC. Metformin has been demonstrated to be an antineoplastic agent in some types of cancer; however, it has not been sufficiently valued in treating GC because the effect of metformin in combination with chemotherapy regimens has not yet been evaluated. The present study aimed to evaluate the mechanisms underlying cell death induced by metformin alone or when combined with chemotherapy. The cytogenetic characteristics of the NCI-N87 cell line were determined by fluorescence in situ hybridization (FISH). To determine viability, the cells were treated with metformin, epirubicin, cisplatin, docetaxel and 5-fluorouracil (individually and at different concentrations). Subsequently, the cells were treated with metformin alone, and in combination with the chemotherapeutic drugs and the epirubicin + cisplatin + 5-fluorouracil, docetaxel + cisplatin + 5-fluorouracil, and cisplatin + 5-fluorouracil regimens. Cell viability, proliferation and mitochondrial membrane potential (ΔΨm) were analyzed by spectrophotometry. Apoptosis, caspase activity and cell cycle progression were assessed by flow cytometry. Finally, light microscopy was used to evaluate senescence and clonogenicity. The results revealed that metformin, alone and when combined with chemotherapy, increased the proportion of apoptotic cells, promoted the loss of ΔΨm, and induced apoptosis through caspase activity in GC cells. Moreover, metformin decreased cell proliferation. In addition, metformin alone did not induce senescence and it counteracted the effects of chemotherapy-induced senescence in GC cells. Additionally, metformin, alone and when combined with chemotherapy, decreased the clonogenic capacity of NCI-N87 GC cells. In conclusion, metformin may increase the effects of chemotherapy on NCI-N87 cell death and could represent an option to improve the treatment of GC.

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.

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.

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.

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.

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.

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.

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.

Detection of monoclonal IGH rearrangements in circulating cells from healthy first-degree relatives of patients with multiple myeloma.

Multiple myeloma (MM) is characterized by abnormal proliferation of clonal plasma cells or monoclonal plasmacytosis, resulting in accumulation of clonal immunoglobulins. Monoclonal gammopathy of unknown significance (MGUS) is considered a premorbid stage for developing MM. Studies have shown an increased risk of MGUS in first-degree relatives of patients with MM. Detection of immunoglobulin heavy chain gene (IGH) rearrangement provides a useful tool for assessing clonality. The aim of this study was to determine clonality in peripheral blood samples from 61 healthy first-degree relatives of MM probands by sorting circulating lymphocytes and detection of the IGH rearrangements in these cells. We detected 16 out of 61 (26.2%) relatives with monoclonal complete and incomplete IGH rearrangements; only three of them showed elevated monoclonal immunoglobulin in the serum protein electrophoresis. We conclude that this strategy is able to identify efficiently clonality in peripheral blood samples from first-degree relatives of patients with MM, who have a non-negligible risk of developing MGUS or other plasma cell dyscrasias.

Gamma-irradiation induced apoptosis in peritoneal macrophages by oxidative stress. Implications of antioxidants in caspase mitochondrial pathway.

The in vivo and in vitro development of apoptosis induced by gamma-irradiation was studied in mouse peritoneal macrophages. The apoptosis index was measured by fluorescence microscopy and DNA electrophoresis. In vivo apoptosis was greatest eight days after 8 Gy total body gamma-irradiation. A DNA ladder electrophoretic pattern was only observed in the gamma-irradiated group. The participation of reactive oxygen species in apoptosis induction was investigated by pretreating mice with the antioxidants superoxide dismutase, catalase, vitamin E or lipopolysaccharide before gamma-irradiation. Measurement of serum lipoperoxides showed oxidative stress in the gamma-irradiated mice and the protection given by the antioxidants. These results were confirmed using in vitro cultures of peritoneal macrophages: gamma-irradiated groups and antioxidant-pretreated gamma-irradiation groups showed results similar to those observed with in vivo irradiation. A loss of mitochondrial membrane potential (delta psi(m)) was also observed by microscopy in the gamma-irradiated cell cultures. Experiments with caspase inhibitors confirmed the participation of caspase 3 and caspase 9.