Implications of NLRP3 Suppression Using Glibenclamide and miR-223 against Colorectal Cancer.

The NLR family pyrin domain containing 3 (NLRP3) promotes the growth of colorectal cancer (CRC). However, the therapeutic effect of NLRP3 inhibition on CRC cell progression is controversial. This study comparatively investigated the therapeutic effect of a pharmacological NLRP3 inhibitor, glibenclamide (gli), and the post-translational suppression of NLRP3 by miR-223 on CRC cell progression in HCT-116 and HCT-15 cells. LPS and ATP were used to activate Gli-treated and LSB-hsa-miR-223-3p (WTmiR-223)-expressing HCT-116 cells. NLRP3.AB.pCCL.sin.cPPT.U6.miR-223-Decoy.hPGK.GFP.WPRE plasmid (DmiR-223) was the negative control for miR-223 expression. NLRP3, gasdermin D, and BAX expressions were analyzed using western blotting. Real-time PCR detected the RNA expression of autophagy-related genes ATG5, BECN1, and miR-223 in non-transfected cells. ELISA analyzed IL-1ß and IL-18 in the medium. MTS-1, annexin V, wound-healing, and sphere-invasion assays were used to assess cell viability and progression. A multiplex cytokine assay detected proinflammatory cytokine secretion. LPS-ATP-activated NLRP3 produced gasdermin D cleavage, released IL-1b and IL-18, and activated cell migration and sphere invasion. In contrast, reduced cell growth, miR-223 expression, IFN-γ, CXCL10, and LIF secretion were found in cells after inflammasome activation. Both gli and WTmiR-223 induced autophagy genes ATG5 and BECN1 and reduced the NLRP3 activation and its downstream proteins. However, while gli had a limited effect on the production of IFN-γ, CXCL10, and LIF, WTmiR-223 increased the release of those cytokines. In addition, gli did not suppress cell growth, while WTmiR-223 promoted apoptosis. Notably, neither gli nor WTmiR-223 effectively prevented sphere invasion. These data suggest that, while WTmiR-223 could have a better anticancer effect in CRC compared to gli, the sole usage of miR-223-mediated NLRP3 suppression may not be sufficient to prevent CRC metastasis.

Leaf Extract from European Olive (Olea europaea L.) Post-Transcriptionally Suppresses the Epithelial-Mesenchymal Transition and Sensitizes Gastric Cancer Cells to Chemotherapy.

The overall survival of patients with the advanced and recurrent gastric cancer (GC) remains unfavorable. In particular, this is due to cancer spreading and resistance to chemotherapy associated with the epithelial-mesenchymal transition (EMT) of tumor cells. EMT can be identified by the transcriptome profiling of GC for EMT markers. Indeed, analysis of the TCGA and GTEx databases (n = 408) and a cohort of GC patients (n = 43) revealed that expression of the CDH2 gene was significantly decreased in the tumors vs. non-tumor tissues and correlated with the overall survival of GC patients. Expression of the EMT-promoting transcription factors SNAIL and ZEB1 was significantly increased in GC. These data suggest that targeting the EMT might be an attractive therapeutic approach for patients with GC. Previously, we demonstrated a potent anti-cancer activity of the olive leaf extract (OLE). However, its effect on the EMT regulation in GC remained unknown. Here, we showed that OLE efficiently potentiated the inhibitory effect of the chemotherapeutic agents 5-fluorouracil (5-FU) and cisplatin (Cis) on the EMT and their pro-apoptotic activity, as was demonstrated by changes in the expression of the EMT markers (E- and N-cadherins, vimentin, claudin-1) in GC cells treated with the aforementioned chemotherapeutic agents in the presence of OLE. Thus, culturing GC cells with 5-FU + OLE or Cis + OLE attenuated the invasive properties of cancer cells. Importantly, upregulation of expression of the apoptotic markers (PARP cleaved form) and increase in the number of cells undergoing apoptosis (annexin V-positive) were observed for GC cells treated with a combination of OLE and 5-FU or Cis. Collectively, our data illustrate that OLE efficiently interferes with the EMT in GC cells and potentiates the pro-apoptotic activity of certain chemotherapeutic agents used for GC therapy.

Co-loading of Temozolomide with Oleuropein or rutin into polylactic acid core-shell nanofiber webs inhibit glioblastoma cell by controlled release.

Glioblastoma (GB) has susceptibility to post-surgical recurrence. Therefore, local treatment methods are required against recurrent GB cells in the post-surgical area. In this study, we developed a nanofiber-based local therapy against GB cells using Oleuropein (OL), and rutin and their combinations with Temozolomide (TMZ). The polylactic acid (PLA) core-shell nanofiber webs were encapsulated with OL (PLAOL), rutin (PLArutin), and TMZ (PLATMZ) by an electrospinning process. A SEM visualized the morphology and the total immersion method determined the release characteristics of PLA webs. Real-time cell tracking analysis for cell growth, dual Acridine Orange/Propidium Iodide staining for cell viability, a scratch wound healing assay for migration capacity, and a sphere formation assay for tumor spheroid aggressiveness were used. All polymeric nanofiber webs had core-shell structures with an average diameter between 133 ± 30.7-139 ± 20.5 nm. All PLA webs promoted apoptotic cell death, suppressed cell migration, and spheres growth (p < 0.0001). PLAOL and PLATMZ suppressed GB cell viability with a controlled release that increased over 120 h, while PLArutin caused rapid cell inhibition (p < 0.0001). Collectively, our findings suggest that core-shell nano-webs could be a novel and effective therapeutic tool for the controlled release of OL and TMZ against recurrent GB cells.

Olea europaea L. Leaf Extract Attenuates Temozolomide-Induced Senescence-Associated Secretion Phenotype in Glioblastoma.

Objectives: The purpose of this study was to investigate the effect of Olea europaea L. leaf extract (OLE) on senescence and senescence-associated secretory phenotype (SASP) caused by temozolomide (TMZ) in glioblastoma (GB). Materials and Methods: A senescence ß-galactosidase assay and a colony formation assay were used to determine the effects of OLE, TMZ, and OLE + TMZ on the cellular senescence and aggressiveness of GB cell lines T98G and U87MG. mRNA expression levels of p53, a senescence factor, interleukin (IL)-6, matrix metalloproteinases (MMP)-9, and nuclear factor kappa B1 (NF-κB1) as SASP factors and Bcl-2 and Bax as senolytic markers were assessed using quantitative reverse transcription-real-time polymerase chain reaction. Cells were double-stained with acridine orange and propidium iodide to observe the cell morphology. Results: TMZ increased the senescence rate of GB cells (p<0.001). Besides, OLE + TMZ reduced the proportion of senescent cells (p<0.001) and their capability to form colonies compared to TMZ-only-treated cells. Additionally, OLE + TMZ co-treatment elevated the mRNA expression levels of MMP-9, IL-6, NF-κB1, p53, and the Bax/Bcl-2 ratio compared to TMZ-only treatment. Especially in U87MG cells, involvement of OLE in TMZ treatments increased more than six times in the Bax/Bcl-2 ratio compared to TMZ-only, which induced the apoptosis-like morphological features (p<0.0001). Conclusion: Collectively, our findings presented the inhibitory effect of OLE on TMZ-mediated SASP-factor production in GB and, accordingly, its potential contribution to elongate the time of recurrence.

Diabetes Mellitus-Mediated MALAT1 Expression Induces Glioblastoma Aggressiveness.

AIM: To describe the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in glioblastoma (GB) progression in patients concurrently diagnosed with diabetes mellitus (DM). MATERIAL AND METHODS: Formalin-fixed paraffin-embedded (FFPE) tumor samples of 47 patients diagnosed with GB only and 13 patients diagnosed with GB and DM (GB-DM) were enrolled in this study. Data for p53 and Ki67 immunohistochemical staining of the tumors and blood HbA1c levels of patients with DM were retrospectively collected. MALAT1 expression was assessed using quantitative real-time polymerase chain reaction. RESULTS: The coexistence of GB and DM induced the nuclear expression of p53 and Ki67 compared with GB only. MALAT1 expression was higher in GB-DM tumors than in GB only tumors. The expression of MALAT1 and HbA1c levels were positively correlated. Additionally, MALAT1 was positively correlated with tumoral p53 and Ki67. The disease-free survival of patients with GB-DM with high MALAT1 expression was shorter than that of those diagnosed with GB only and with a lower MALAT1 expression. CONCLUSION: Our findings suggest that one of the mechanisms of the facilitating effect of DM on GB tumor aggressiveness is via MALAT1 expression.

Blocking the Hormone Receptors Modulates NLRP3 in LPS-Primed Breast Cancer Cells.

NOD-like receptor protein 3 (NLRP3) may contribute to the growth and propagation of breast cancer (BC). The effect of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) on NLRP3 activation in BC remains unknown. Additionally, our knowledge of the effect of blocking these receptors on NLRP3 expression is limited. We used GEPIA, UALCAN, and the Human Protein Atlas for transcriptomic profiling of NLRP3 in BC. Lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP) were used to activate NLRP3 in luminal A MCF-7 and in TNBC MDA-MB-231 and HCC1806 cells. Tamoxifen (Tx), mifepristone (mife), and trastuzumab (Tmab) were used to block ER-α, PR, and HER2, respectively, on inflammasome activation in LPS-primed MCF7 cells. The transcript level of NLRP3 was correlated with ER-ɑ encoding gene ESR1 in luminal A (ER-α+, PR+) and TNBC tumors. NLRP3 protein expression was higher in untreated and LPS/ATP-treated MDA-MB-231 cells than in MCF7 cells. LPS/ATP-mediated NLRP3 activation reduced cell proliferation and recovery of wound healing in both BC cell lines. LPS/ATP treatment prevented spheroid formation in MDA-MB-231 cells but did not affect MCF7. HGF, IL-3, IL-8, M-CSF, MCP-1, and SCGF-b cytokines were secreted in both MDA-MB-231 and MCF7 cells in response to LPS/ATP treatment. Tx (ER-α inhibition) promoted NLRP3 activation and increased migration and sphere formation after LPS treatment of MCF7 cells. Tx-mediated activation of NLRP3 was associated with increased secretion of IL-8 and SCGF-b compared to LPS-only-treated MCF7 cells. In contrast, Tmab (Her2 inhibition) had a limited effect on NLRP3 activation in LPS-treated MCF7 cells. Mife (PR inhibition) opposed NLRP3 activation in LPS-primed MCF7 cells. We have found that Tx increased the expression of NLRP3 in LPS-primed MCF7. These data suggest a link between blocking ER-α and activation of NLRP3, which was associated with increased aggressiveness of the ER-α+ BC cells.

Olea europaea Leaf Phenolics Oleuropein, Hydroxytyrosol, Tyrosol, and Rutin Induce Apoptosis and Additionally Affect Temozolomide against Glioblastoma: In Particular, Oleuropein Inhibits Spheroid Growth by Attenuating Stem-like Cell Phenotype.

The effects of Olea europaea leaf extract (OLE) phenolics, including oleuropein (OL), hydroxytyrosol (HT), tyrosol (TYR), and rutin against glioblastoma (GB), independently and in combination with temozolomide (TMZ), were investigated in T98G and A172 cells. Cell growth was assessed by WST-1, real-time cell analysis, colony formation, and cell cycle distribution assays. A dual acridine orange propidium iodide (AO/PI) staining and annexin V assay determined cell viability. A sphere-forming assay, an intracellular oxidative stress assay, and the RNA expression of CD133 and OCT4 investigated the GB stem-like cell (GSC) phenotype. A scratch wound-healing assay evaluated migration capacity. OL was as effective as OLE in terms of apoptosis promotion (p < 0.001) and GSC inhibition (p < 0.001). HT inhibited cell viability, GSC phenotype, and migration rate (p < 0.001), but its anti-GB effect was less than the total effect of OLE alone. Rutin decreased reactive oxygen species production and inhibited colony formation and cell migration (p < 0.001). TYR demonstrated the least effect. The additive effects of OL, HT, TYR and rutin with TMZ were significant (p < 0.001). Our data suggest that OL may represent a novel therapeutic approach against GB cells, while HT and rutin show promise in increasing the efficacy of TMZ therapy.

Azithromycin and Ceftriaxone Differentially Activate NLRP3 in LPS Primed Cancer Cells.

BACKGROUND: Cancer patients are prescribed antibiotics, such as macrolides and lactamides, for infection treatment. However, the effect of these antibiotics on NLRP3 activation remains largely unknown. METHOD: Lung cancer (A549) and prostate cancer (PC3) cell lines were primed with lipopolysaccharide (LPS) to activate NLRP3 transcription. Cells were then treated with azithromycin (Az) or ceftriaxone (Cf). NLRP3 activation was analyzed by qPCR, Western blot, and ELISA. Cell growth and viability were assessed by real-time cell analysis and Annexin V expression. Levels of 41 cytokines were also analyzed using a multiplex assay. RESULTS: LPS-Az activated transcription of NLRP3, Pro-CASP-1, and Pro-IL-1ß in A549 cells, while failing to upregulate NLRP3 and Pro-IL-1ß in PC3 cells. LPS-Az decreased the secretion of pro-inflammatory cytokines while it induced the pro-angiogenic factors in A549 and PC3 cells. In contrast, LPS-Cf suppressed the expression of NLRP3-associated genes, NLRP3 protein expression, the inflammatory cytokine secretion in A549 and PC3 cells. LPS-Az and LPS-Cf had a limited effect on cell growth and viability. DISCUSSION: Our data suggest that Cf could suppress LPS induced NLRP3, which should be considered when selecting antibiotics for cancer treatment. In contrast, the effect of Az on LPS primed NLRP3 and the inflammatory cytokines production appears to depend on the cancer cell origin. Therefore, these data indicate that considerations are required when selecting Az for the treatment of cancer patients.

Early-stage colon cancer with high MALAT1 expression is associated with the 5-Fluorouracil resistance and future metastasis.

BACKGROUND: This study aimed to investigate the role of long noncoding RNA (LncRNA) expression profiles to predict relapse and 5-FU response in patients with stage I/II colon cancer (CC). METHODS AND RESULTS: The expression level of 15 LncRNA was analyzed in stage I/II colon tumors of 126 CC patients. To confirm the findings in-vitro, 5FU-resistant HT29 cells were generated by subjecting HT-29 cells to the increasing concentrations of 5FU for 6 months. The 5FU resistance was observed in WST-1 and Annexin V analyses. The colony formation and wound healing assays were assessed to determine the metastatic properties of the cells. Expression levels of LncRNAs and mRNA of EMT-related genes were determined by RT-PCR. The role of LncRNA on metastasis and 5FU sensitivity were confirmed in pcDNA3.0-PTENP1 and si-MALAT1 expressed 5FU-resistant HT29 cell lineages. RESULTS: High MALAT1 (p = 0.0002) and low PTENP1 (p = 0.0044) expressions were significantly associated with 5-FU resistance and tumor relapse in stage I/II CC. The invasiveness and colony-forming characteristics of 5-FU-resistant cell lineages were higher as compared to the parent HT-29. Moreover, the expression of MALAT1 (p = 0.0009) was increased while the expression of PTENP1 (p = 0.0158) decreased in 5FU-resistant-HT-29 cells. Si-MALAT1 treatment increased cell sensitivity to 5FU, whereas it decreased invasive behaviors of 5 FU-resistant-HT-29 cells. CONCLUSION: MALAT1 may be a biomarker in predicting recurrence in early-stage CC. Our findings suggest that a cell-based therapy to target MALAT1 could be established for these patients to prevent metastasis and 5-FU resistance.

The Age-Dependent Role of Th22, Tc22, and Tc17 Cells in the Severity of Pneumonia in COVID-19 Immunopathogenesis.

Coronavirus disease 2019 (COVID-19) has clinical manifestations ranging from mild symptoms to respiratory failure, septic shock, and multi-organ failure. Lymphocytes are divided into different subtypes based on their cytokine production pattern. In this study, we investigated the role of cytokine expressions of CD4+ T (T helper [Th]1, Th2, Th17, Th22) and CD8+ T cell subtypes (T cytotoxic [Tc]1, Tc2, Tc17, Tc22) in the pathogenesis of COVID-19. Peripheral blood mononuclear cells (PBMCs) were extracted with Ficoll by density gradient centrifugation from blood samples of 180 COVID-19 patients (children and adults) and 30 healthy controls. PBMCs were stimulated with PMA and Ionomycin and treated with Brefeldin A in the fourth hour, and a 10-colored monoclonal antibody panel was evaluated at the end of the sixth hour using flow cytometry. According to our findings, the numbers of Th22 (CD3+, CD4+, and interleukin [IL]-22+) and Tc22 (CD3+, CD8+, IL-22+) cells increased in adult patients regardless of the level of pneumonia (mild, severe, or symptom-free) as compared with healthy controls (p < 0.05). In addition, the number of Tc17 (CD3+, CD8+, and IL-17A+) cells increased in low pneumonia and severe pneumonia groups compared with the healthy controls (p < 0.05). Both IL-22 and IL-17A production decreased during a follow-up within 6 weeks of discharge. Our findings suggest that the increase in only IL-22 expressed Tc22 cells in the 0-12 age group with a general symptom-free course and higher levels of Th22 and Tc22 in uncomplicated adult cases may indicate the protective effect of IL-22. On the contrary, the association between the severity of pneumonia and the elevation of Tc17 cells in adults may reveal the damaging effect of IL-22 when it is co-expressed with IL-17.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases.

Doxycycline Attenuates Cancer Cell Growth by Suppressing NLRP3-Mediated Inflammation.

NLR family pyrin domain containing 3 (NLRP3) inflammasome formation is triggered by the damaged mitochondria releasing reactive oxygen species. Doxycycline was shown to regulate inflammation; however, its effect on NLRP3 in cancer remains largely unknown. Therefore, we sought to determine the effect of doxycycline on NLRP3 regulation in cancer using an in vitro model. NLRP3 was activated in a prostate cancer cell line (PC3) and a lung cancer cell line (A549) before treatment with doxycycline. Inflammasome activation was assessed by analyzing RNA expression of NLRP3, Pro-CASP-1, and Pro-IL1ß using RT-qPCR. Additionally, NLPR3 protein expression and IL-1ß secretion were analyzed using Western blot and ELISA, respectively. Tumor cell viability was determined using Annexin V staining and a cell proliferation assay. Cytokine secretion was analyzed using a 41Plex assay for human cytokines. Data were analyzed using one-way ANOVA model with Tukey's post hoc tests. Doxycycline treatment decreased NLRP3 formation in PC3 and A549 cells compared to untreated and LPS only treated cells (p < 0.05). Doxycycline also decreased proliferation and caused cell death through apoptosis, a response that differed to the LPS-Nigericin mediated pyroptosis. Our findings suggest that doxycycline inhibits LPS priming of NLRP3 and reduces tumor progression through early apoptosis in cancer.

DPYD c.1905 + 1G>A Promotes Fluoropyrimidine-Induced Anemia, a Prognostic Factor in Disease-Free Survival, in Colorectal Cancer.

Background and Aim: In 10-30% of colorectal cancer (CRC) patients, toxic reactions occur after fluoropyrimidine-based chemotherapy. A dihydropyridine dehydrogenase (DPYD) gene variant, c.1905 + 1G>A, leads to intolerance to fluoropyrimidines. Due to the low frequency of this variant in many populations, the prevalence of fluoropyrimidine-induced hematologic side effects in CRC patients with the c.1905 + 1G>A variant is unclear. In this study, we investigated the prevalence of the DPYD c.1905 + 1 variants in a Turkish CRC cohort and the potential effects of these variants on fluoropyrimidine-induced hematologic side effects. Materials and Methods: The DPYD c.1905 + 1 variant was genotyped using polymerase chain reaction-restriction fragment length polymorphism analysis and confirmed by Sanger sequencing in peripheral blood samples of 100 CRC patients who received fluoropyrimidine-based chemotherapy and 60 healthy volunteers. The association of c.1905 + 1 variants with susceptibility to hematologic side effects was evaluated. Results: The DPYD c.1905 + 1G>A variant was more common in the CRC group than in the healthy control group (p = 0.001). The presence of the c.1905 + 1G>A variant was associated with thrombocytopenia (p = 0.039) and anemia (p = 0.035). CRC patients with fluoropyrimidine-induced anemia had shorter disease-free survival than CRC patients without fluoropyrimidine-induced anemia (p = 0.0009). Conclusions: Before administering fluoropyrimidine-based chemotherapy, genetic screening for the DPYD c.1905 + 1G>A variant should be performed with the aim of preventing anemia and anemia-induced complications in CRC patients.

Rab GTPase Mediating Regulation of NALP3 in Colorectal Cancer.

The NALP3 inflammasome signaling contributes to inflammation within tumor tissues. This inflammation may be promoted by the vesicle trafficking of inflammasome components and cytokines. Rab5, Rab7 and Rab11 regulate vesicle trafficking. However, the role of these proteins in the regulation of inflammasomes remains largely unknown. To elucidate the role of these Rab proteins in inflammasome regulation, HCT-116, a colorectal cancer (CRC) cell line expressing pDsRed-Rab5 wild type (WT), pDsRed-Rab5 dominant-negative (DN), pDsRed-Rab7 WT, pDsRed-Rab7 DN, pDsRed-Rab11 WT and pDsRed-Rab11 DN were treated with lipopolysaccharide (LPS)/nigericin. Inflammasome activation was analyzed by measuring the mRNA expression of NLRP3, Pro-CASP1, RAB39A and Pro-IL-1ß, conducting immunofluorescence imaging and western blotting of caspase-1 and analysing the secretion levels of IL-1ß using enzyme-linked immunosorbent assay (ELISA). The effects of Rabs on cytokine release were evaluated using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-Premixed 41 Plex. The findings showed that LPS/nigericin-treated cells expressing Rab5-WT indicated increased NALP3 expression and secretion of the IL-1ß as compared to Rab5-DN cells. Caspase-1 was localized in the nucleus and cytosol of Rab5-WT cells but was localized in the cytosol in Rab5-DN cells. There were no any effects of Rab7 and Rab11 expression on the regulation of inflammasomes. Our results suggest that Rab5 may be a potential target for the regulation of NALP3 in the treatment of the CRC inflammation.

Roles of novel IL-1 family (IL-36, IL-37, and IL-38) members in chronic brucellosis.

The secretion of interleukin (IL)-1 family cytokines is one of the most potent and earliest pro-inflammatory responses triggered by brucellosis. However, the roles of the most recently discovered IL-1 family members, IL-36, IL-37, and IL-38, in the transition into the chronic form of brucellos is remain largely unknown. Therefore, in this study, the roles of IL-36, IL-37, and IL-38 in brucella infections and their effects on the transition from the acute to chronic form of the disease were investigated. Using peripheral blood samples from 40 patients with acute brucellosis, 40 patients with chronic brucellosis, and 40 healthy control subjects, we analysed the serum concentrations of secreted IL-36, IL-37, and IL-38 using ELISA. The findings were confirmed by using RT-qPCR to analyse the mRNA levels of the genes encoding IL-36, IL-37, and IL-38 in peripheral blood mononuclear cells (PBMCs) from 10 randomly selected patients from each of the three groups. Our results showed that serum IL-37 (p < 0.001) and IL-38 (p < 0.001) concentrations were lower in patients with brucellosis than in the healthy controls. In addition, serum IL-37 and IL-38 concentrations were higher in the chronic patient group than in the acute patient group. The mRNA expression levels of IL-37 and IL1F10, genes that encode IL-38, did not affect serum cytokine secretion levels. This result suggests that the high secretion levels of IL-37 and IL-38 may be related to the progression into the chronic form of brucellosis. Our findings will aid in clarifying the mechanism of the transition of brucellosis from the acute to the chronic form of the disease.

Therapeutic Potential of Pharmacological Targeting NLRP3 Inflammasome Complex in Cancer.

Introduction: Dysregulation of NLRP3 inflammasome complex formation can promote chronic inflammation by increased release of IL-1ß. However, the effect of NLRP3 complex formation on tumor progression remains controversial. Therefore, we sought to determine the effect of NLRP3 modulation on the growth of the different types of cancer cells, derived from lung, breast, and prostate cancers as well as neuroblastoma and glioblastoma in-vitro. Method: The effect of Caspase 1 inhibitor (VX765) and combination of LPS/Nigericin on NLRP3 inflammasome activity was analyzed in A549 (lung cancer), MCF-7 (breast cancer), PC3 (prostate cancer), SH-SY5Y (neuroblastoma), and U138MG (glioblastoma) cells. Human fibroblasts were used as control cells. The effect of VX765 and LPS/Nigericin on NLRP3 expression was analyzed using western blot, while IL-1ß and IL-18 secretion was detected by ELISA. Tumor cell viability and progression were determined using Annexin V, cell proliferation assay, LDH assay, sphere formation assay, transmission electron microscopy, and a multiplex cytokine assay. Also, angiogenesis was investigated by a tube formation assay. VEGF and MMPs secretion were detected by ELISA and a multiplex assay, respectively. Statistical analysis was done using one-way ANOVA with Tukey's analyses and Kruskal-Wallis one-way analysis of variance. Results: LPS/Nigericin increased NRLP3 protein expression as well as IL-1ß and IL-18 secretion in PC3 and U138MG cells compared to A549, MCF7, SH-SY5Y cells, and fibroblasts. In contrast, MIF expression was commonly found upregulated in A549, PC3, SH-SY5Y, and U138MG cells and fibroblasts after Nigericin treatment. Nigericin and a combination of LPS/Nigericin decreased the cell viability and proliferation. Also, LPS/Nigericin significantly increased tumorsphere size in PC3 and U138MG cells. In contrast, the sphere size was reduced in MCF7 and SH-SY5Y cells treated with LPS/Nigericin, while no effect was detected in A549 cells. VX765 increased secretion of CCL24 in A549, MCF7, PC3, and fibroblasts as well as CCL11 and CCL26 in SH-SY5Y cells. Also, VX765 significantly increased the production of VEGF and MMPs and stimulated angiogenesis in all tumor cell lines. Discussion: Our data suggest that NLRP3 activation using Nigericin could be a novel therapeutic approach to control the growth of tumors producing a low level of IL-1ß and IL-18.

Long noncoding RNA MALAT1 may be a prognostic biomarker in IDH1/2 wild-type primary glioblastomas.

Primary glioblastoma (GB) is the most aggressive type of brain tumors. While mutations in isocitrate dehydrogenase (IDH) genes are frequent in secondary GBs and correlate with a better prognosis, most primary GBs are IDH wild-type. Recent studies have shown that the long noncoding RNA metastasis associated lung adenocarcinoma transcript-1 (MALAT1) is associated with aggressive tumor phenotypes in different cancers. Our aim was to clarify the prognostic significance of MALAT1 in IDH1/2 wild-type primary GB tumors. We analyzed IDH1/2 mutation status in 75 patients with primary GB by DNA sequencing. The expression of MALAT1 was detected in the 75 primary GB tissues and 5 normal brain tissues using reverse transcription quantitative PCR (RT-qPCR). The associations between MALAT1 expression, IDH1/2 mutation status, and clinicopathological variables of patients were determined. IDH1 (R132H) mutation was observed in 5/75 primary GBs. IDH2 (R172H) mutation was not detected in any of our cases. MALAT1 expression was significantly upregulated in primary GB vs. normal brain tissues (p = 0.025). Increased MALAT1 expression in IDH1/2 wild-type primary GBs correlated with patient age and tumor localization (p = 0.032 and p = 0.025, respectively). A multivariate analysis showed that high MALAT1 expression was an unfavorable prognostic factor for overall survival (p = 0.034) in IDH1/2 wild-type primary GBs. High MALAT1 expression may have a prognostic role in primary GBs independent of IDH mutations.

MicroRNA Post-transcriptional Regulation of the NLRP3 Inflammasome in Immunopathologies.

Inflammation has a crucial role in protection against various pathogens. The inflammasome is an intracellular multiprotein signaling complex that is linked to pathogen sensing and initiation of the inflammatory response in physiological and pathological conditions. The most characterized inflammasome is the NLRP3 inflammasome, which is a known sensor of cell stress and is tightly regulated in resting cells. However, altered regulation of the NLRP3 inflammasome is found in several pathological conditions, including autoimmune disease and cancer. NLRP3 expression was shown to be post-transcriptionally regulated and multiple miRNA have been implicated in post-transcriptional regulation of the inflammasome. Therefore, in recent years, miRNA based post-transcriptional control of NLRP3 has become a focus of much research, especially as a potential therapeutic approach. In this review, we provide a summary of the recent investigations on the role of miRNA in the post-transcriptional control of the NLRP3 inflammasome, a key regulator of pro-inflammatory IL-1ß and IL-18 cytokine production. Current approaches to targeting the inflammasome product were shown to be an effective treatment for diseases linked to NLRP3 overexpression. Although utilizing NLRP3 targeting miRNAs was shown to be a successful therapeutic approach in several animal models, their therapeutic application in patients remains to be determined.

Prediction of breast cancer metastasis risk using circulating tumor markers: A follow-up study.

Distant organ tumor dissemination is a major cause of breast cancer-related deaths. In 2010, we analyzed the prognostic importance of the circulating tumor markers (CTMs) cytokeratin 19 (CK19), CK20, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in relation to the clinical and pathological characteristics of patients with breast cancer (BC). To assess the clinical utility of CK19, CK20 and EGFR in predicting distant metastasis in BC, here we report 7-year follow-up results of 77 patients. The patients with at least one positive CTM were classified as CTM(+) and those negative for all CTMs were assigned to CTM(-) group. In patients who received no treatment following CTM analysis, 25.0% had metastasis in CTM(+) and 10.0% in CTM(-) group. In patients who received one of the following therapies: chemotherapy, radiotherapy or hormone therapy, or the combinations of these therapies, the rate of metastasis was 33.3% in CTM(+) and 20.0% in CTM(-) group. Disease-free time was shorter in CTM(+) patients compared to CTM(-) group (28.83 ± 10.76 and 41.38 ± 9.5 months, respectively). According to multivariate Cox proportional hazard regression analysis, the presence of regional lymph node metastasis, Ki-67 expression, higher tumor grade and CTM expression status were predictors of poor prognosis associated with distant metastasis (p < 0.05). Also, CTM positivity was a factor associated with metastasis-related poor prognosis (HR = 0.492, p = 0.026). The mean survival for CTM(+) patients was shorter than that for CTM(-) patients (90.671 ± 2.66 and 101.23 ± 3.92 months, respectively; p > 0.05). Our findings demonstrate that CTM positivity may indicate a high metastasis risk; however, CTM negativity does not guarantee low metastasis risk. These results may encourage further preclinical investigation of CTMs, to evaluate the possible implications of these findings to the clinical setting.

The microRNA expression signature of CD4+ T cells in the transition of brucellosis into chronicity.

Brucellosis is a serious infectious disease that continues to be a significant cause of morbidity worldwide and across all ages. Despite early diagnosis and treatment, 10-30% of patients develop chronic brucellosis. Although there have been recent advances in our knowledge of Brucella virulence factors and hosts' immune response to the infection, there is a lack of clear data regarding how the infection bypasses the immune system and becomes chronic. The present study investigated immunological factors and their roles in the transition of brucellosis from an acute to a chronic infection in CD4+ T cells. CD4+ T cells sorted from peripheral blood samples of patients with acute or chronic brucellosis and healthy controls using flow cytometry as well as more than 2000 miRNAs were screened using the GeneSpring GX (Agilent) 13.0 miRNA microarray software and were validated using reverse transcription polymerase chain reaction (RT-qPCR). Compared to acute cases, the expression levels of 28 miRNAs were significantly altered in chronic cases. Apart from one miRNA (miR-4649-3p), 27 miRNAs were not expressed in the acute cases (p <0.05, fold change> 2). According to KEGG pathway analysis, these miRNAs are involved in the regulation of target genes that were previously involved in the MAPK signalling pathway, regulation of the actin cytoskeleton, endocytosis, and protein processing in the endoplasmic reticulum. This indicates the potential role of these miRNAs in the development of chronic brucellosis. We suggest that these miRNAs can be used as markers to determine the transition of the disease into chronicity. This is the first study of miRNA expression that analyses human CD4+ T cells to clarify the mechanism of chronicity in brucellosis.