Abstract
Trillium tschonoskii Maxim (TTM), a traditional Chinese medicine, has been demonstrated to have a potent anti-tumor effect. Recently, polyphyllin VI (PPVI), a main saponin isolated from TTM, was reported by us to significantly suppress the proliferation of non-small cell lung cancer (NSCLC) via the induction of apoptosis and autophagy in vitro and in vivo. In this study, we further found that the NLRP3 inflammasome was activated in PPVI administrated A549-bearing athymic nude mice. As is known to us, pyroptosis is an inflammatory form of caspase-1-dependent programmed cell death that plays an important role in cancer. By using A549 and H1299 cells, the in vitro effect and action mechanism by which PPVI induces activation of the NLRP3 inflammasome in NSCLC were investigated. The anti-proliferative effect of PPVI in A549 and H1299 cells was firstly measured and validated by MTT assay. The activation of the NLRP3 inflammasome was detected by using Hoechst33324/PI staining, flow cytometry analysis and real-time live cell imaging methods. We found that PPVI significantly increased the percentage of cells with PI signal in A549 and H1299, and the dynamic change in cell morphology and the process of cell death of A549 cells indicated that PPVI induced an apoptosis-to-pyroptosis switch, and, ultimately, lytic cell death. In addition, belnacasan (VX-765), an inhibitor of caspase-1, could remarkably decrease the pyroptotic cell death of PPVI-treated A549 and H1299 cells. Moreover, by detecting the expression of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD in A549 and h1299 cells using Western blotting, immunofluorescence imaging and flow cytometric analysis, measuring the caspase-1 activity using colorimetric assay, and quantifying the cytokines level of IL-1β and IL-18 using ELISA, the NLRP3 inflammasome was found to be activated in a dose manner, while VX-765 and necrosulfonamide (NSA), an inhibitor of GSDMD, could inhibit PPVI-induced activation of the NLRP3 inflammasome. Furthermore, the mechanism study found that PPVI could activate the NF-κB signaling pathway via increasing reactive oxygen species (ROS) levels in A549 and H1299 cells, and N-acetyl-L-cysteine (NAC), a scavenger of ROS, remarkably inhibited the cell death, and the activation of NF-κB and the NLRP3 inflammasome in PPVI-treated A549 and H1299 cells. Taken together, these data suggested that PPVI-induced, caspase-1-mediated pyroptosis via the induction of the ROS/NF-κB/NLRP3/GSDMD signal axis in NSCLC, which further clarified the mechanism of PPVI in the inhibition of NSCLC, and thereby provided a possibility for PPVI to serve as a novel therapeutic agent for NSCLC in the future.
Highlights
Lung cancer, as one of the most common malignant tumors, is the leading cause of cancer-related death worldwide [1]
Through further detection of the NLRP3 inflammasome in the tumor tissue of A549-bearing athymic nude mice using Western blotting and immunohistochemistry methods, Figure 1B showed that polyphyllin VI (PPVI) significantly improved the protein expression of NLRP3, cleaved-caspase-1, cleaved-IL-1β and cleaved-Gasdermin D (GSDMD) in tumor tissue
To further validate the correlation of cell death with the activated NLRP3 inflammasome, as well as the increased reactive oxygen species (ROS) level in PPVI-treated A549 and H1299 cells, we found that NAC could reverse NLRP3 expression and cell death induced by PPVI
Summary
As one of the most common malignant tumors, is the leading cause of cancer-related death worldwide [1]. It has a dismal prognosis and a five year survival rate lower than 15% [2]. Non-small cell lung cancer (NSCLC), which includes squamous cell carinoma, adenocarcinoma and large-cell carcinoma, is a classic subtype of lung cancer and accounts for 85% of all lung cancer cases [3]. There are many therapeutic methods, including surgery, radiotherapy and chemotherapy, chemotherapy is still the common strategy for lung cancer treatment [4,5]. Chemotherapy is less sensitive to NSCLC as compared to small lung cancer [6]. Novel strategies are essential for the improvement of clinical survival rate based on a better understanding of tumor biology
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