Therapeutic Potential of Pharmacological Targeting NLRP3 Inflammasome Complex in Cancer

Abstract

Introduction: Dysregulation of NLRP3 inflammasome complex formation can promote chronic inflammation by increased release of IL-1β. 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-1β 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-1β 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-1β and IL-18.

Keywords: IL-1β; NLRP3; cancer; inflammasome; nigericin.

Figure 1

Effect of NLRP3 modulation in U138MG and SH-SY5Y cells, where one of the highest and one of the lowest NLRP3 expression was observed and in Fibroblasts: Nigericin (20 µM, Invivogen) treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml, Sigma, St. Louis, USA) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM, Invivogen). (A–C) NLRP3 protein expression was demonstrated by western blot. (D–F) IL-1β and IL-18 levels were quantified, from inside the cells and in the medium representing levels secreted from the cells, by ELISA. the cells by ELISA. U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. *p < 0.05, n = 3.

Figure 2

Effect of NLRP3 inhibition and stimulation on cell viability and proliferation of PC3, SH-SY5Y cells, and Fibroblasts. Nigericin (20 µM), treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml), was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM). (A–C) Expression of Annexin V (D, F, H) LDH cytotoxicity assay and (E, G, I) a real-time cell proliferation assay. U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. *p < 0.05, n = 3.

Figure 3

Effect of NLRP3 modulation on sphere formation of tumor cell lines. Nigericin (20 µM) treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM). U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. *p < 0.05, n = 3.

Figure 4

Cytokine release patterns in A549, MCF7, PC3, SH-SY5Y, U138MG cells, and fibroblasts after NLRP3 modulation. Nigericin (20 µM), treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM). Camptothecin (6 µM, Sigma) treatment of cells for 24 h was used to induce apoptosis. U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. N = 3.

Figure 5

The effect of NLRP3 inflammasome modulation on cell morphology in A549, PC3, and human fibroblast cells. Nigericin (20 µM, Invivogen) treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml, Sigma, St. Louis, USA) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM, Invivogen). (A) In untreated PC3 cells, small vacuoles were found in the cytoplasm, some of them contain electron-dense material. The cytoplasm is electron-dense containing many free ribosomes. (B) In untreated A549 cells, ER appears as narrow, elongated, rough tanks with a large number of free ribosomes. Also, oval-shaped mitochondria with clear cristae and an average electron density matrix are visualized. (C) In untreated fibroblasts, free ribosomes and polyribosomes were found in the cytoplasm. The shape of mitochondria was elongated with a condensed matrix and a large number of cristae. VX765 treatment: (D) significantly affected the ultrastructure of PC3 cells; increased numbers of large membrane-bound vacuoles were found in the cytoplasm, where some of them were merged or contained multi-vesicular aggregates. There was no visible difference in the ultrastructure of mitochondria between VX765 treated and untreated PC3 cell mitochondria. Cristae were slightly clearer compared to untreated cells. (E) In A549 cells, increased numbers of free ribosomes and granularization of ER were detected. Mitochondria were slightly enlarged and the outer membrane of mitochondria and their cristae became clear. (F) in fibroblasts, increased numbers of organelles, granularization of ER and changes to the ER structure to an expanded, branched, and irregular shape was identified. There were few free ribosomes and many polyribosomes. Additionally, the Golgi apparatus was expanded with a large number of vesicles. Mitochondria displayed a round or oval shape with condensed matrix and lacked distinct cristae. LPS treatment: (G) In PC3 cells, an increased number of organelles was identified. ER cisternae were expanded. The Golgi apparatus developed cistern stacks. Functional activity of mitochondria was increased: the ultrastructure of mitochondria was changed: the length of the mitochondria was considerably elongated, the matrix became electron-dense, and some of the cristae were visible. (H) In A549 cells, after LPS treatment; ER cisternae were expanded, had an irregular shape and formed a network. (I) In fibroblasts, similar to the effect of VX765; after LPS treatment protein synthesis and export were considerably increased; where ER cisternae were greatly expanded which had an irregular shape and formed a network. Additionally, the Golgi apparatus was visualized with well-developed cisternae stacks and a large number of vesicles in the cytoplasm. These vesicles were also detected near the cytoplasmic membrane of the cells; some of them were involved in exocytosis. After Nigericin or combined LPS and Nigericin treatments: (J, M, K, N). PC3 and A549 cells showed signs of lytic cell death such as nuclear condensation, numerous round transparent vacuoles in the cytoplasm, pore formation in the cell membrane, cell swelling and bursting. The morphology of the mitochondria was significantly changed as compared to control where: the shape of the organelle was toroidal; the matrix became electron-dense with few elongated cristae or, the absence of cristae. (L). Different from the tumor cell lines, in Fibroblasts, Nigericin treatment did not cause death cell morphology. Nigericin affected the nucleus structure where the integrity of the karyolemma appeared slightly destroyed. Mitochondria were round or oval, with clear cristae. The cytoplasm contained few free ribosomes and many polyribosomes. (O). After combined LPS and Nigericin treatment fibroblasts demonstrated a lytic cell death morphology including nuclear condensation, large electron-transparent vacuoles and pore formation in the cell membrane, cell swelling and bursting.

Figure 6

The effect of NLRP3 inflammasome modulation on Δψm in tumor cell lines and human fibroblasts. Nigericin (20 µM, Invivogen) treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml, Sigma, St. Louis, USA) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM, Invivogen). U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. *P < 0.001, **P < 0.05.

Figure 7

The effects of releasing cytokines from PC3 cells after inhibition or stimulation of the NLRP3 inflammasome on the HUVEC tube formation. Nigericin (20 µM) treatment for 24 h with and without 3 h pre-incubation with LPS (1 µg/ml) was used to activate the NLRP3 inflammasome. To inhibit Caspase 1, cells were treated with VX765 (20 µM). U, Untreated; V, VX765; L, LPS; N, Nigericin; LN, LPS/Nigericin. *p<0.05, n=3.