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.

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.

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.

A Comparative Study Between Hybrid Abutments and Standard Abutments in Implant-Supported Prosthesis: A Split-Mouth Clinical Trial.

Background Implant-supported prostheses are widely used to replace extracted teeth. Therefore, studies on abutments' designs, shapes, and benefits had increased in recent years, as the design of the standard abutment still poses many problems in periodontal and cosmetic aspects. So, could the hybrid abutment solve some of these problems? Aim We aim to conduct a clinical comparison between standard and hybrid abutments in terms of the state of peri-implant gingival tissues and patients' aesthetic and functional satisfaction after the cementation of the final prostheses. Material and methods The study sample consisted of 10 patients, with 20 dental implants. Each patient received two implants as a standard abutment was placed over one implant and a hybrid abutment was placed over the other. Clinical assessment of the peri-implant gingival tissue and patients' aesthetic and functional satisfaction was performed (immediately, three months, six months, and one year) after the cementation of the final prostheses. The Mann-Whitney U test was used to detect statistically significant differences between groups. Results The percentage of the thick gingival biotype was 80%, and the percentage of the thin gingival biotype was 20% in each group during the follow-up periods. In addition, all papilla fill the whole interdental space in all samples of the two groups after six months and one year. Finally, there were no significant differences in patients' aesthetic satisfaction between groups during one year of follow-up (P = 0.631), and there were no significant differences in patients' functional satisfaction between groups during one year of follow-up (P = 0.684). Conclusion Within the limitations of the current work, there are no differences between standard and hybrid abutments in terms of affecting the peri-implant gingival tissue and patients' aesthetic and functional satisfaction.

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.

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.