Chemotherapeutic drugs cause increased release of pro-inflammatory molecules from MM cells.

<p>(A) Hmeso MM cells treated with either Dox or cisplatin caused increased release of HMGB1, FGF2, IL-1β and IL-18 as measured by Western blot analysis or ELISA. No loading controls were included as analysis was performed in cell culture medium. (B) H2373 MM cells also showed increased release of various pro-inflammatory molecules in response to drugs. IL-6 levels were either decreased (H2373) or not significantly changed (Hmeso) by drug treatments. *p≤0.05 as compared to untreated controls (0).</p

Combination of IL-1R antagonist and cisplatin attenuated MM tumor weight/volume in an intraperitoneal xenograft mouse model.

<p>SCID mice injected with Hmeso MM cells received either cisplatin (2mg/kg, ip, 1X/week for 2 weeks), Anakinra (92 mg/kg, ip, 2x daily for 3 weeks), Cisplatin and Anakinra together or saline. (A, B) tumor weights and volumes were drastically reduced in combination group. (C) Total cell count was not significantly affected by combination treatment. (D) Neutrophil counts were significantly lower in cisplatin and combination group. *p≤0.05 as compared to saline treated group; †p≤0.05 as compared to cisplatin alone (by Student’s‘t’ test).</p

MM cells and tumors have low levels of NLRP3 protein and caspase-1 activity.

<p>A. Seven human MM cell lines were assessed for NLRP3 steady-state mRNA levels and compared with human mesothelial cell line (LP9), *p≤0.05 as compared to MM cell lines; B. NLRP3 protein levels in 4 human MM cell lines as compared to LP9 (red-NLRP3 protein, green-nucleus); C. NLRP3 mRNA levels in human MM tumor tissues (T) and normal counter parts (N); D. NLRP3 protein levels in human MM tumor tissue arrays as compared to normal lung (red-NLRP3 protein, green-nucleus); E. Caspase-1 activity in human MM cell lines as compared to LP9, *p≤0.05 as compared to MM cell lines. Scale bar = 50 microns.</p

Chemotherapeutic drugs cause priming and activation of the NLRP3 inflammasome in MM cell lines.

<p>Treatment of Hmeso MM cells with Dox or cisplatin resulted in increased NLRP3 protein levels (A) and activation of inflammasome (B). Activation was measured by increased caspase-1 p20 release into the medium. *p≤0.05 as compared to untreated controls (0). (C) Cisplatin increased the protein levels of NLRP3 in H2373 MM cells and both Dox and cisplatin caused activation of caspase-1 in H2373 cells as measured by caspase-1 p20 release into the medium (D). *p≤0.05 as compared to untreated controls.</p

Chemotherapeutic drugs increase NLRP3 levels in human MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM,) for 24 or 48 h and steady-state NLRP3 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</p

Chemotherapeutic drugs regulate PYCARD/ASC and pro-caspase levels in MM cell lines.

<p>Human MM cell lines were treated with different concentrations (0–100 μM) of cisplatin or Dox (0–25 μM) for 24 or 48 h and steady-state PYCARD/ASC or pro-caspase-1 mRNA levels were assessed by qRT-PCR. *p≤0.05 as compared to untreated (0) controls at the same time point.</p

Chemotherapeutic drugs cause pyroptosis in MM cell lines.

<p>MTS assay performed to measure cell viability on Hmeso or H2373 MM cells in presence of Dox or cisplatin with and without specific caspase-1 inhibitor (A, B) or pan caspase inhibitor (C, D). The effect of these inhibitors on drug-induced caspase-1 activity was also performed in both MM cell lines (E, F). *p≤0.05 as compared to untreated controls (0); †p≤0.05 as compared to similar treatment without caspase- inhibitor, ‡ p≤0.05 as compared to low dose caspase inhibitor.</p