Doxycycline Attenuates Cancer Cell Growth by Suppressing NLRP3-Mediated Inflammation

Mohammad Alsaadi, Gulcin Tezcan, Ekaterina E Garanina, Shaimaa Hamza, Alan McIntyre, Albert A Rizvanov, Svetlana F Khaiboullina, Mohammad Alsaadi, Gulcin Tezcan, Ekaterina E Garanina, Shaimaa Hamza, Alan McIntyre, Albert A Rizvanov, Svetlana F Khaiboullina

Abstract

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-IL1β using RT-qPCR. Additionally, NLPR3 protein expression and IL-1β 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.

Keywords: apoptosis; cancer; doxycycline; inflammasome; nod-like receptor protein 3 (NLRP3).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The experimental design. (A) The effect of LPS, Nigericin and Glibenclamide on NLRP3 activation. LPS as the signal 1 is a priming trigger inducing the NLRP3 and pro-IL-1β production. Nigericin is the signal 2 and plays role in polymerization of an active NLRP3 inflammasome complex which recruits pro-Caspase-1 and cleaved it to active form of Caspase-1. The Caspase-1 liberates functional IL-1β and IL-18, regulatory cytokines of inflammation. Glibenclamide is a potassium channel inhibitor and blocks the polymerization of NLRP3 protein complex. (B) The experimental control groups consisted of: 1. A549 and PC3 cells treated with glibenclamide for 24 h as the positive control of NLRP3 inhibition; 2. LPS-only for 3 h as the positive control of NLRP3 priming; 3. LPS+nigericin treatment for 24 h as the positive control of active NLRP3 inflammasome complex. The experimental test groups consisted of A549 and PC3 cells treated with doxycycline-only for 24 h and pretreated with LPS for 3 h. U: Untreated PC3 or A549 cells, G: Glibenclamide, L: LPS, N: Nigericin, D: Doxycycline, PRR: The cytosolic pattern recognition receptor, AC1: Active Caspase-1, PC1: Pro-Caspase-1, ASC: Apoptosis-associated speck-like protein containing a CARD, ROS: reactive oxygen species.
Figure 2
Figure 2
The effect of doxycycline on NLRP3 inflammasome activation. Doxycycline effects on: (A–C) The RNA levels of NLRP3 downstream genes in PC3 (A–C), and A549 cells (D–F). The effect of doxycycline on NLRP3 protein translation (G), cell-secretion of IL-1β in PC3 (H), and A549 cells (I). U: Untreated, G: Glibenclamide, L: LPS, LN: LPS-Nigericin, D: Doxycycline, LD: LPS Doxycycline. * adjusted p < 0.05 compared to untreated cells; ◊: adjusted p < 0.05 compared to LN treated cells. Data were analyzed using one-way ANOVA and Tukey’s post hoc tests (n = 3).
Figure 3
Figure 3
The effect of doxycycline mediated inhibition of NLRP3 on proliferation and viability of cancer cells. (A) Doxycycline and LPS-doxycycline effect on PC3 cell proliferation; (B) Doxycycline and LPS-doxycycline effect on A549 cell proliferation; (C) PC3 cell viability, (D) A549 cell viability. The adjusted p-values were calculated using one-way ANOVA and Tukey’s post hoc tests. U: Untreated, L: LPS, D: Doxycycline, LD: LPS-Doxycycline. * p < 0.05 compared to untreated cells ◊ p < 0.05 compared to LPS treated cells; n = 3.
Figure 4
Figure 4
Effect of Doxycycline on cytokine release pattern in PC3 and A549 cells. (A,B). The heat map graphs of cytokine release pattern in PC3 and A549 cells. (B,C). The time-dependent secretion of IL-1β. (D–H). The time-dependent secretion of EGF and GM-CSF in PC3 and A549 cells. U: Untreated, G: Glibenclamide, L: LPS, LN: LPS-Nigericin, D: Doxycycline, LD: LPS-Doxycycline.

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