Natural Product Triptolide Induces GSDME-mediated Pyroptosis In Head And Neck Cancer Through Suppressing Mitochondrial Hexokinase-Ⅱ

【Objective】Pyroptosis is a lytic cell death form executed by Gasdermin family proteins.Induction of tumor pyroptosis promotes anti-tumor immunity and is a potential cancer treatment strategy.Triptolide(TPL)is a natural product isolated from the traditional Chinese herb which possesses potent anti-tumor activity in human cancers.However,the mechanism by which it eliminates tumor remains to be elucidated.This study intends to deeply study the anti-tumor role of natural product triptolide in the treatment of head and neck cancer,and to analyze the mechanism of the key mitochondrial hexokinase-Ⅱ between aerobic glycolysis and anti-tumor activity of TPL.【Methods】In this study,the effect of TPL on the survival of head and neck cancer cells HK1,FaDu and C666-1 were measured by MTT and colony formation assays.Cell apoptosis was determined by Annexin Ⅴ/PI assay.Different cell death pathways inhibitors,such as Fer-1,DFOM,3-MA,CQ,Nec-1 were used in combination with TPL to treat head and neck cancer cells,the cell morphology was observed by microscopy and cell viability was detected by MTT.Pyroptosis was evaluated through observing the morphological characteristics of cells by microscope,as well as detecting the release of interleukin-1β in culture mediums by ELISA,and measuring the activity of LDH by colorimetry.The expression of Gasdermins genes in HK1,FaDu and C666-1 cells were detected by RT-PCR assays and verified by Western blot.In HK1 and FaDu cells,shRNA expressing lentivirus was used to stably interfere with the expression of GSDME and HK-Ⅱ,and qRT-PCR and Western blot were respectively verified.HK1 and FaDu cells were treated with Z-VAD,a pan-caspase inhibitor of caspases,in combination with TPL.The cell morphology was observed under microscope,and the activation of Caspase 3 and the cleavage of GSDME were detected by Western blot.Cell immunofluorescence staining was used to analyze the effect of TPL on the expression and localization of GSDME,c-Myc,NRF2 and SLC7A11.The expression of Bad,Bax,cytochrome c and HK-Ⅱ in mitochondria and cytoplasmic proteins were detected by subcellular fraction assays.RNA-Seq was used to determine the transcriptomes of two TPL-treated nasopharyngeal carcinoma cell lines HK1 and C666-1 to study TPL induced transcriptomic alterations.Differentially expressed genes regulated by TPL were analyzed by bioinformatics integration,then verified by qRT-PCR,Western blot and immunofluorescence experiments,respectively.Cycloheximide,an inhibitor of protein synthesis,was used to examine the effect of TPL treatment on the half-life of c-Myc protein.Using proteasome inhibitors MG132 and PS341,the effect of TPL on post-translational modification of c-Myc was investigated.Mitochondria of living cells were stained with Mito Tracker Red FM,and the binding of HK-Ⅱ protein to mitochondria was detected by immunofluorescence assays.The metabolic levels of HK1,FaDu and C666-1 cells were measured by detected glucose consumption,lactate production and cellular ATP content following TPL treatment.The DCFHDA probe detected the expression level of ROS in TPL-treated cells.GEPIA database and UALCAN database were used to analyze the expression of GSDME in head and neck squamous cancer cell.Using nasopharyngeal carcinoma cells HK1-LV16,a nude mice subcutaneous xenograft tumor model was established and treated by intraperitoneal injection of TPL or Erastin.The transplanted tumor was subjected to H&E staining and immunohistochemical detection.【Results】In this study,we demonstrated that TPL significantly inhibited the cell survival of head and neck cancer cells HK1 and FaDu in a dose-dependent manner,while the survival of C666-1 cells was very little affected by TPL.This result suggested that HK1 and FaDu cells were sensitive to TPL,while C666-1 cells were resistant to TPL.However,Annexin V/PI staining revealed that TPL treatment did not cause apoptosis in head and neck cancer cells.In addition,using different cell death pathway inhibitors in combination with TPL to treat head and neck cancer cells,we found that TPL-induced cell death may not be ferroptosis,autophagy and necroptosis.HK1,FaDu cells treated with TPL displayed robust cell death,cytoplasmic swelling and membrane rupture,dramatic release of IL-1β and LDH into the culture mediums.Morphological and biochemical features clearly indicated that TPL selectively induced pyroptosis in head and neck cancer cells HK1 and FaDu.Notably,TPL did not induce pyroptosis in C666-1 cells despite decreased cell confluency upon TPL treatment.On mechanism,TPL suppresses head and neck cancer cells malignant behaviors through inducing Gasdermin E(GSDME)mediated pyroptosis.Silencing GSDME attenuates the cytotoxicity of TPL against cancer cells.TPL promotes mitochondrial translocation of Bad and Bax proteins,leading to release of cytochrome c to cytoplasm and subsequent cleavage of GSDME by active Caspase 3.GSEA analysis indicated that genes downregulated by TPL were functionally enriched in processes related to c-Myc targets and glycolysis.TPL simultaneously inhibits protein stability and transcription of c-Myc in head and neck cancer cells.RNA-Seq indicated that hexokinase II(HK-Ⅱ),which is a known target gene of c-Myc that catalyzes the first rate-limiting step of glycolysis,was down-regulated by TPL.TPL inhibited aerobic glycolysis of cancer cells,as evidenced by reduced glucose consumption,lactate production and cellular ATP content following TPL treatment.Stable depletion of HK-Ⅱ was not sufficient to trigger pyroptosis of cancer cells,but promoted TPL induced pyroptosis.In contrast,forced expression of HK-Ⅱ prevented pyroptosis induced by TPL in HK1 and FaDu cells.TPL treatment activated Bad/Bax-Caspase3-GSDME cascade through repressing mitochondrial associated HK-Ⅱ.Furthermore,TPL treatment suppresses NRF2/SLC7A11(also known as x CT)axis regardless of the level of GSDME expression in the cells,resulting in excessive accumulation of reactive oxygen species(ROS)in head and neck cancer cells.TPL was not enough to induce ferroptosis,it increased the sensitivity of tumor cells to Erastin,an inhibitor of SLC7A11,which can induce ferroptosis.It is worthy noting that either TPL or erastin alone exerted little effects on survival of C666–1 cells,but these two drugs synergistically killed the C666–1 cancer cell.TPL has an antitumor effect on head and neck cancer in nude mice model,and the combination of TPL and Erastin exerts a strong synergistic effect on tumor inhibition.Based on the above research,we think that combination of TPL with Erastin is a promising strategy for head and neck cancer therapy.This study not only provides a new paradigm of TPL in anti-tumor activity,but also highlights the crucial role of mitochondrial HK-Ⅱ in the connection between glucose metabolism and pyroptosis.【Conclusions】1.TPL eliminates head and neck cancer cells through inducing pyroptotic cell death rather than apoptosis.2.TPL induces cancer cells pyroptosis is mediated by GSDME.Mechanistically,TPL treatment represses c-Myc/HK-Ⅱ axis and activates Bad/Bax-Caspase 3-GSDME cascade through repressing mitochondrial associated HK-Ⅱ in head and neck cancer cells.3.TPL simultaneously inhibites NRF2/SLC7A11 axis and induces reactive oxygen species(ROS)accumulation.Although TPL was not enough to induce ferroptosis,it increased the sensitivity of tumor cells to Erastin,an inhibitor of SLC7A11,to induce ferroptosis.4.TPL has anti-tumor effect on head and neck cancer in nude mice model,and synergistic effect with Erastin can improve the killing effect on tumor.