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Implications of NLRP3 Suppression Using Glibenclamide and miR-223 against Colorectal Cancer
Shaimaa Hamza1, Ekaterina E Garanina1, Layaly Shkair1
1Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008 Kazan, Russia.
Abstract:
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-1β 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.
Insights
This study compared glibenclamide and miR-223 for NLRP3 inhibition in colorectal cancer (CRC). miR-223 showed better anti-cancer effects than glibenclamide but neither fully prevented metastasis.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- The NLR family pyrin domain containing 3 (NLRP3) inflammasome is implicated in colorectal cancer (CRC) progression.
- The therapeutic efficacy of NLRP3 inhibition in CRC remains debated, necessitating comparative studies.
Purpose of the Study:
- To comparatively investigate the therapeutic effects of pharmacological NLRP3 inhibition (glibenclamide) versus post-translational suppression (miR-223) on CRC cell progression.
- To assess the impact of these interventions on NLRP3 activation, downstream signaling, and key cellular processes like apoptosis and invasion.
Main Methods:
- Utilized HCT-116 and HCT-15 CRC cell lines, activating NLRP3 with LPS and ATP.
- Administered glibenclamide (pharmacological inhibitor) and WTmiR-223 (miR-223 expression) or DmiR-223 (control).
- Analyzed protein expression (NLRP3, gasdermin D, BAX), RNA expression (autophagy genes, miR-223), cytokine levels (IL-1β, IL-18, IFN-γ, CXCL10, LIF), cell viability, apoptosis, migration, and invasion.
Main Results:
- NLRP3 activation induced gasdermin D cleavage, IL-1β/IL-18 release, and promoted cell migration/invasion.
- Both glibenclamide and WTmiR-223 reduced NLRP3 activation and downstream proteins, inducing autophagy genes ATG5 and BECN1.
- WTmiR-223 demonstrated superior effects by promoting apoptosis and increasing pro-inflammatory cytokines (IFN-γ, CXCL10, LIF) compared to glibenclamide, though neither prevented sphere invasion.
Conclusions:
- miR-223-mediated NLRP3 suppression exhibits a potentially greater anti-cancer effect in CRC than glibenclamide.
- While miR-223 enhances apoptosis and certain cytokine profiles, it does not fully inhibit CRC cell invasion.
- Targeting NLRP3 solely via miR-223 may not be sufficient to prevent colorectal cancer metastasis, suggesting combination therapies might be necessary.
