BackgroundNecroptotic susceptibility is probably an intrinsic weakness of cancer. Here, we report that resibufogenin, a member of bufadienolide family, suppresses the growth and metastasis of colorectal cancer (CRC) through induction of necroptosis in vivo.MethodsSW480 cells with stably expressing enhanced green fluorescence protein were xenografted to BALB/c-nu mice to observe the growth of tumors. Liver metastasis was observed by injection of MC38 cells beneath the splenic capsule of mice. Protein expression was determined by immunohistochemistry, immunofluorescence and western blot.ResultsConsolidated in vitro results indicate that resibufogenin has anti-proliferative activity on CRC cells. PI staining and transmission electron microscope imaging suggest that the cell death induced by resibufogenin are mainly through necrosis, which is further confirmed by the ineffectiveness of z-VAD, a pan-caspase general inhibitor. In particular, resibufogenin induced necrosis is substantially abrogated in receptor-interacting protein kinase 3 (RIPK3) knockout mouse embryo fibroblasts. The RIP3-dependent necrosis has been classified as necroptosis. Resibufogenin triggeres necroptosis through upregulating RIP3 and phosphorylating mixed lineage kinase domain-like protein at Ser358. Resibufogenin also activates the expression of PYGL, GLUD1 and GLUL in a RIP3-dependent manner. Resibufogenin exerts cytotoxic effect by inducing reactive oxygen species accumulation which can be neutralized by N-acetylcysteine. Remarkably, resibufogenin significantly suppresses liver-metastasis from spleen implantation. The anti-neoplastic effect of this compound can be abrogated by RIP3 knockdown.ConclusionResibufogenin suppresses growth and metastasis of CRC through RIP3-mediated necroptosis.Electronic supplementary materialThe online version of this article (10.1186/s12967-018-1580-x) contains supplementary material, which is available to authorized users.

Inducing angiogenesis is a hallmark of cancers that sustains tumor growth and metastasis. Neovascularization is a surprisingly early event during the multistage progression of cancer. Cinobufagin, an important bufadienolide originating from Chan Su, has been clinically used to treat cancer in China since the Tang dynasty. Here, we show that cinobufagin suppresses colorectal cancer ( CRC ) growth in vivo by downregulating angiogenesis. The hierarchized neovasculature is significantly decreased and the vascular network formation is disrupted in HUVEC by cinobufagin in a dose‐dependent way. Endothelial apoptosis is observed by inducing reactive oxygen species ( ROS ) accumulation and mitochondrial dysfunction which can be neutralized by N‐acetyl‐ l ‐cysteine ( NAC ). Expression of hypoxia‐inducible factor 1α ( HIF ‐1α) is reduced and phosphorylation of mTOR at Ser2481 and Akt at Ser473 is downregulated in HUVEC . Endothelial apoptosis is triggered by cinobufagin by stimulation of Bax and cascade activation of caspase 9 and caspase 3. Increased endothelial apoptosis rate and alterations in the HIF ‐1α/ mTOR pathway are recapitulated in tumor‐bearing mice in vivo. Further, the anti‐angiogenesis function of cinobufagin is consolidated based on its pro‐apoptotic effects on an EOMA ‐derived hemangioendothelioma model. In conclusion, cinobufagin suppresses tumor neovascularization by disrupting the endothelial mTOR / HIF ‐1α pathway to trigger ROS ‐mediated vascular endothelial cell apoptosis. Cinobufagin is a promising natural anti‐angiogenetic drug that has clinical translation potential and practical application value.

Colorectal cancer is generally believed to progress through an adenoma - carcinoma sequence. Adenomatous polyposis coli (APC) mutations serve as the initiating event in adenoma formation. The Apc mouse harbors a mutation in the APC gene, which is similar or identical to the mutation found in individuals with familial adenomatous polyposis and 70% of all sporadic CRC cases. Autophagy is a constitutive process required for proper cellular homeostasis. However, its role in intestinal adenoma formation is still controversial. Atractylenolide I (AT1) is a sesquiterpenoid that possesses various clinically relevant properties such as anti-tumor and anti-inflammatory activities. The role of AT1 on adenoma formation was tested in Apc mice and its underlying mechanism in regulating autophagy was documented. D-dopachrome tautomerase (D-DT) was identified as a potential target of AT1 by an proteomics-based approach. The effects of p53 modification on autophgic flux was monitored in p53 and p53 HCT116 cells. Small interfering RNA was used to investigate the function of Atg7 and D-DT on autophagy programme induce by AT1. AT1 effectively reduced the formation of adenoma and downregulated the tumorigenic proteins in Apc mice. Importantly, AT1 stimulated autophagic flux through downregulating acetylation of p53. Activation of Sirt1 by AT1 was essential for the deacetylation of p53 and downregulation of D-DT. The lowered expression of COX-2 and β-catenin by AT1 were partly recovered by Atg7 knockdown. AT1 activates autophagy machinery to downregulate D-DT and reduce intestinal adenoma formation. This discovery provides evidence in vivo and in vitro that inducing autophagy by natural products maybe a potential therapy to ameliorate colorectal adenoma formation.

Obesity increases the risk of colorectal cancer (CRC) by 30%. The obese tumor microenvironment compromises antitumor immunity by eliciting exhausted T cells (Tex). Hypothesizing that Dahuang Fuzi Baijiang decoction (DFB) is a combined classical prescription from the “Synopsis of Prescriptions of the Golden Chamber”. We first determined that DFB regresses tumor growth in high‐fat diet–induced obese mice by expanding the TIM3− subset with intermediate expression of programmed cell death‐1 (PD‐1intTIM3−) and restricting the PD‐1hiTIM3+ subset. Transcription factor 1 (TCF1) is highly expressed in the PD‐1intTIM3− subset but is absent in PD‐1hiTIM3+ cells. We next confirmed that progenitor PD‐1intTCF+ cells robustly produce tumor necrosis factor‐α (TNFα) and interferon‐γ, whereas terminally differentiated PD‐1intTCF+ cells have defects in generating TNFα. With transgenic ob/ob mice, we found that DFB produces cooperative efficacy with anti‐PD‐1 (αPD‐1) by limiting the PD‐1hiTim3+ subset and amplifying the PD‐1intTCF+ population. Finally, we defined the recombinant chemokine C‐C‐motif receptor 2 (CCR2)+CD8+ subset as terminal Tex and identified that the differentiation from progenitor to terminal Tex is driven, at least in part, by the chemokine (C‐C motif) ligand 2 (CCL2)/CCR2 axis. The CCR2 inhibitor enhances the response to αPD‐1 by promoting the counts of progenitor Tex. Altogether, DFB dampens CCL2 and preserves progenitor Tex in the obese microenvironment to restrain CRC progression. These findings provide unambiguous evidence that the traditional Chinese formula DFB can prevent tumor progression by modulating adaptive immunity and establish a strong rationale for further clinical verification.