Natural product triptolide induces GSDME-mediated pyroptosis in head and neck cancer through suppressing mitochondrial hexokinase-ΙΙ

Abstract

Background: Pyroptosis is a lytic cell death form executed by gasdermins 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, its role in pyroptosis remains to be elucidated.

Methods: Cell survival was measured by colony formation assay. Cell apoptosis was determined by Annexin V assay. Pyroptosis was evaluated by morphological features and release of interleukin 1β and lactate dehydrogenase A (LDHA). Immunofluorescence staining was employed to measure subcellular localization of proteins. Tumorigenicity was assessed by a xenograft tumor model. Expression levels of mRNAs or proteins were determined by qPCR or western blot assay, respectively.

Results: Triptolide eliminates head and neck cancer cells through inducing gasdermin E (GSDME) mediated pyroptosis. Silencing GSDME attenuates the cytotoxicity of TPL against cancer cells. TPL treatment suppresses expression of c-myc and mitochondrial hexokinase II (HK-II) in cancer cells, leading to activation of the BAD/BAX-caspase 3 cascade and cleavage of GSDME by active caspase 3. Silencing HK-II sensitizes cancer cells to TPL induced pyroptosis, whereas enforced expression of HK-II prevents TPL induced pyroptosis. Mechanistically, HK-II prevents mitochondrial translocation of BAD, BAX proteins and activation of caspase 3, thus attenuating cleavage of GSDME and pyroptosis upon TPL treatment. Furthermore, TPL treatment suppresses NRF2/SLC7A11 (also known as xCT) axis and induces reactive oxygen species (ROS) accumulation, regardless of the status of GSDME. Combination of TPL with erastin, an inhibitor of SLC7A11, exerts robust synergistic effect in suppression of tumor survival in vitro and in a nude mice model.

Conclusions: This study not only provides a new paradigm of TPL in cancer therapy, but also highlights a crucial role of mitochondrial HK-II in linking glucose metabolism with pyroptosis.

Keywords: Ferroptosis; Gasdermins; Head and neck squamous cell carcinoma; Nasopharyngeal carcinoma; X(c)(−) cysteine/glutamate antiporter.

Fig. 1

TPL induces pyroptosis in head and neck cancer cells. a Cell viability assays. HK1, FaDu and C666–1 cells were exposed to various concentrations of TPL (0, 5, 25, 50 and 150 nM) for 24 h or 48 h. Cell viability were determined by MTT assays. b Colony formation assays. Cells were treated with various doses of TPL (0, 1, 2, and 5 nM) for 48 h and then allowed to grow for 2 weeks in fresh culture medium. Colonies were visualized by crystal purple staining. Values are mean ± SD from triplicate experiments. c Apoptotic cell frequencies treated without or with TPL (50 nM) for 24 h were determined by annexin V/PI assays. d Morphological alterations induced by TPL (50 nM) treatment. Arrow showed cell swelling and rupture. e IL-1β released into culture medium was detected by ELISA assays. f LDH1 activity assays. LDH1 activity in culture mediums of cells treated without or with TPL (50 nM) for 24 h were measured by LDH1 kit. g Release of LDHA proteins to culture mediums were detected by western blot assays. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

TPL induced pyroptosis in head and neck cancer cells is executed by GSDME. a The mRNA levels of gasdermin members were determined by RT-PCR assays. b The protein levels of GSDME were measured by western blot assays. c The Protein levels of full-length GSDME and GSDME-N terminus in cells treated without or with TPL (50 nM) for 24 h were measured by western blot assays. d GSDME proteins localization in cells treated without or with TPL (50 nM) for 24 h were visualized by immunofluorescence assays. e The mRNA or protein levels of GSDME in shRNAs expressing lentivirus infected cells were measured by qPCR or western blot. f Morphological features of cells treated with TPL showed reduced pyroptotic cell death in GSDME-silenced cells. Cells were treated with TPL (50 nM) for 24 h. g Cell viability was measured by MTT assay. h Cell survival was measured by colony formation assay. Cells were treated with TPL (10 nM) for 24 h. i LDH1 activity assays. j IL-1β released into culture medium was detected by ELISA assays. Values are shown as mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Activation of mitochondrial pathway and caspase 3 are required for cleavage of GSDME and pyroptosis induced by TPL. a The protein levels of mitochondrial death pathway were determined by western blot. b sub-cellular distributions of BAX, BAD and cytochrome c proteins were determined by subcellular fraction and western blot assays. c The mRNA and protein levels of BAX, BAD in siRNAs transfected cells were determined by qPCR or western blot. d Cleavage of caspase 3 and GSDME in BAX/BAD-depleted cells were measured by western blot. e Morphological features showed less cell swelling and rupture induced by TPL when caspase activity was blocked by z-VAD. f Effects of z-VAD on cleavage of GSDME was measured by western blot. g The mRNA and protein levels of caspase 3 in siRNAs transfected cells were determined by qPCR or western blot. h Cleavage of GSDME in caspase 3 silenced cells were determined by western blot

Fig. 4

TPL suppresses mitochondrial HK-II and glycolysis in head and neck cancer cells. a GSEA analysis indicated TPL induced transcriptomic alterations were associated with c-myc targets and glycolysis. b qPCR assay showed that TPL suppressed c-myc, HK-II mRNA levels in head and neck cancer cells in a dose-dependent manner. c The protein levels of HK-II were measured by western blot. d distribution of HK-II upon TPL treatment was determined by subcellular fraction and western blot. e co-localization of HK-II with mitochondria was assessed by immunofluorescence assay. f glycolytic rates were assessed by glucose consumptions, lactate productions and cellular ATP contents assays. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Mitochondrial HK-II prevents TPL induced pyroptosis. a The mRNA and protein levels of HK-II in shRNAs or cDNA expressing lentivirus infected cells were assessed by qPCR or western blot. b Phase contrast images showed morphological features HK-II silenced or HK-II overexpressed cells upon TPL treatment. c Subcellular fraction assays suggested that silencing HK-II promoted mitochondrial translocation of BAX, BAD. d Silencing HK-II facilitated activation of caspase 3 and cleavage of GSDME upon TPL treatment, whereas overexpression of exogenous HK-II exerted opposite effects. e ELISA assays showed silencing HK-II promoted release of IL-1β into extracellular space, whereas overexpression of exogenous HK-II reduced the release of IL-1β upon TPL treatment. f LDH1 activity assays. g cell survival of HK-II depleted or HK-II overexpressed cells were assessed by colony formation assays. Values are shown as mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

TPL suppresses NRF2/SLC7A11 axis and synergizes with erastin to eliminate both GSDME-expressing and GSDME-deficient cancer cells. a qPCR assays showed TPL suppressed the mRNA levels of NRF2 and SLC7A11 in a dose dependent manner. b The protein levels of NRF2 and SLC7A11 were measured by western blot. c Immunofluorescence assays of NRF2 and SLC7A11 proteins. Cells were treated with or without 50 nM TPL in the absence or presence of erastin (10 μM) for 24 h. d Intracellular ROS were probed with DCFH-DA and measured by FACS. e Phase contrast images showed morphological features of cells under indicated conditions. f cell survival was assessed by colony formation assays. Data are shown as mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control

Fig. 7

Combination of TPL and erastin potently inhibits tumorigenicity of HK1 cell in vivo. a Growth curve of xenograft tumors in mice treated with single reagent or combination of TPL (0.1 mg/kg QD) with erastin (10 mg/kg QD) (n = 5/group). b Xenograft tumors in live mice were visualized in vivo bioluminescence imaging system. c The macroscopic view of mice and xenograft tumors at the endpoint of experiment. d The average tumor weight from different groups. Values are shown as mean ± SD (n = 5). e H&E staining of xenograft tissue sections. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 μm

Fig. 8

TPL represses the protein levels of Ki67, c-myc, HK-II, NRF2 and SLC7A11 in vivo. The expression levels of Ki67, c-myc, HK-II, NRF2 and SLC7A11 proteins in xenograft tumors were measured by IHC staining. Scale bar, 50 μm

Fig. 9

Schematic diagram of mechanisms underlying TPL induced pyroptosis in head and neck cancer cells. TPL simultaneously repressed c-myc/HK-II axis and NRF2/SLC7A11 axis in head and neck cancer cells. Inhibition of mitochondrial HK-II resulted in mitochondrial translocation of BAX and BAD, leading to release of cytochrome c to cytoplasm and subsequent activation of caspase 3. In GSDME ^high cancer cells, active caspase 3 cleaved GSDME at internal linker to liberate the pore-forming activity of GSDME-N, resulting in pyroptosis in cancer cells. At the same time, inhibition of NRF2/SLC7A11 by TPL led to accumulation of ROS in cancer cells, thus making cancer cells were sensitive to cytotoxicity of combination of TPL and erastin, even in GSDME^low cancer cell