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Regressing the accelerated degradation of skeletal muscle protein is a significant goal for cancer cachexia management. Here, we show that genetic deletion of Pgam5 ameliorates skeletal muscle atrophy in various tumor-bearing mice. pgam5 ablation represses excessive myoblast mitophagy and effectively suppresses mitochondria meltdown and muscle wastage. Next, we define BNIP3 as a mitophagy receptor constitutively associating with PGAM5. bnip3 deletion restricts body weight loss and enhances the gastrocnemius mass index in the age- and tumor size-matched experiments. The NH2-terminal region of PGAM5 binds to the PEST motif-containing region of BNIP3 to dampen the ubiquitination and degradation of BNIP3 to maintain continuous mitophagy. Finally, we identify S100A9 as a pro-cachectic chemokine via activating AGER/RAGE. AGER deficiency or S100A9 inhibition restrains skeletal muscle loss by weakening the interaction between PGAM5 and BNIP3. In conclusion, the AGER-PGAM5-BNIP3 axis is a novel but common pathway in cancer-associated muscle wasting that can be targetable. Abbreviation: AGER/RAGE: advanced glycation end-product specific receptor; BA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; Ckm-Cre: creatinine kinase, muscle-specific Cre; CM: conditioned medium; CON/CTRL: control; CRC: colorectal cancer; FUNDC1: FUN14 domain containing 1; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; S100A9: S100 calcium binding protein A9; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TIMM23: translocase of inner mitochondrial membrane 23; TSKO: tissue-specific knockout; VDAC1: voltage dependent anion channel 1.
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BACKGROUND: Sirtuin 3 (Sirt3) is a controversial regulator of carcinogenesis. It residents in the mitochondria and gradually decays during aging. In this study, we tried to investigate the role of Sirt3 in carcinogenesis and to explore its involvement in metabolic alteration. METHODS: We generated conditional intestinal epithelium Sirt3-knockout mice by crossing ApcMin/+; Villin-Cre with Sirt3fl/fl (AVS) mice. The deacetylation site of Lon protease-1 (LONP1) was identified with Mass spectrometry. The metabolic flux phenotype was determined by Seahorse bioanalyzer. RESULTS: We found that intestinal epithelial cell-specific ablation of Sirt3 promotes primary tumor growth via stabilizing mitochondrial LONP1. Notably, we newly identified that Sirt3 deacetylates human oncogene LONP1 at N terminal residue lysine 145 (K145). The LONP1 hyperacetylation-mutant K145Q enhances oxidative phosphorylation to accelerate tumor growth, whereas the deacetylation-mutant K145R produces calorie-restriction like phenotype to restrain tumorigenesis. Sirt3 deacetylates LONP1 at K145 and subsequently facilitates the ESCRT0 complex sorting and K63-ubiquitination that resulted in the degradation of LONP1. Our results sustain the notion that Sirt3 is a tumor-suppressor to maintain the appropriate ubiquitination and degradation of oncogene LONP1. CONCLUSION: Sirt3 represents a targetable metabolic checkpoint of oncogenesis, which produces energy restriction effects via maintaining LONP1 K145 deacetylation and subsequent K63 ubiquitination.
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Neoplasias , Protease La , Sirtuína 3 , Animais , Humanos , Camundongos , Acetilação , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , Transformação Celular Neoplásica , Proteínas Mitocondriais/genética , Protease La/genética , Protease La/metabolismo , Sirtuína 3/metabolismo , UbiquitinaçãoRESUMO
Toads have high medicinal value and have been used for medicinal purposes since the Tang Dynasty period (7th-10th Century AD). Bufarenogin, an active anti-tumor constituent of toad venom, shows anti-tumor activity. In this study, we investigated the inhibitory effects of bufarenogin on the growth and metastasis of colorectal cancer (CRC), particularly its effects on mediating intrinsic signaling pathways that initiate apoptosis. An orthotopic CRC model was established in nude mice via surgical orthotopic implantation to investigate tumor growth. Immunohistochemistry, immunofluorescence, and Western blotting assays were performed to evaluate protein expression. The in vitro results revealed the anti-proliferative effect of bufarenogin against CRC cells. Bufarenogin caused cell death via apoptosis, as revealed by Annexin V/7-amino-actinomycin D double staining, which was verified using a pan-caspase inhibitor. Bufarenogin induced B-cell lymphoma 2-associated X protein (Bax)-dependent intrinsic apoptosis, as demonstrated by mitochondrial translocation of Bax and cytoplasm release of HCT116 wild-type cells and cytochrome C (soluble pro-apoptotic factors). Additionally, we showed that adenine-nucleotide translocator interacted with Bax. Bufarenogin induced intrinsic apoptosis through the cooperation of Bax and adenine-nucleotide translocator and inhibited the metastasis and growth of orthotopical CRC cells.
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Translocador 1 do Nucleotídeo Adenina/metabolismo , Antineoplásicos/uso terapêutico , Bufanolídeos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bufanolídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína X Associada a bcl-2/genéticaRESUMO
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.
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Inibidores da Angiogênese/administração & dosagem , Bufanolídeos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Bufanolídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Transplante de Neoplasias , Fosforilação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) are pervasive transcripts that play pivotal roles in regulating chromatin dynamics, gene and protein expression. Aberrant expression and mutations of lncRNAs represent a driving force behind tumor invasion and metastasis, making them attractive cancer targets. However, most of the lncRNAs are still being discovered and conclusive experimental evidence for their functional relevance is still lacking for most malignancies. In this study, a differentially expressed lncRNA, designated as lnc-CRCMSL, is identified by microarray-based screenings on non-metastatic and metastatic CRC specimens. Lnc-CRCMSL is verified as an anti-metastatic gene and negatively correlated with the poor prognosis of CRC patients. Lnc-CRCMSL overexpression restricts tumor growth and metastasis in vivo and in vitro. Instead, lnc-CRCMSL silencing accelerates CRC cell proliferation and migration. RNA-pulldown assay identifies high mobility group box 2 (HMGB2) as a downstream protein of lnc-CRCMSL. Mechanically, lnc-CRCMSL physically binds to HMGB2 and stabilizes the localization of HMGB2 in the cytoplasm. Notably, lnc-CRCMSL knockdown lead to the shift of HMGB2 into nuclear, in which it triggers epithelial to mesenchymal transition (EMT) programming. Importantly, lnc-CRCMSL controls the cytoplasmic retention of HMGB2 and attenuates the interaction between HMGB2 and OCT4 to suppress EMT. Treatment of leptomycin B (LMB), a potent and specific nuclear export inhibitor, counteracts lnc-CRCMSL-mediated suppression of aggressive phenotypes and EMT process by accumulating the nuclear HMGB2. CONCLUSION: Our data highlight the anti-metastatic role of lnc-CRCMSL in stabilizing HMGB2 through lncRNA-protein interactions in the cytoplasm, and suggest that targeting lnc-CRCMSL may represent a therapeutic opportunity for managing metastatic CRC.
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Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteína HMGB2/genética , Transporte Proteico/genética , RNA Longo não Codificante/genética , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/fisiologia , Proliferação de Células/genética , Citoplasma/fisiologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
BACKGROUND: Necroptotic 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. METHODS: SW480 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. RESULTS: Consolidated 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. CONCLUSION: Resibufogenin suppresses growth and metastasis of CRC through RIP3-mediated necroptosis.
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Apoptose/efeitos dos fármacos , Bufanolídeos/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/ultraestrutura , Humanos , Camundongos , Necrose , Metástase NeoplásicaRESUMO
BACKGROUND: Previous studies showed sanguinarine induced apoptosis in CRC cells but did not define the underlying mechanisms. The purpose of this work was to determine the in vivo and in vitro effects of sanguinarine on CRC tumors and to elucidate the mechanism in regulating the intrinsic apoptosis. METHODS: Cell viability of CRC cell lines treated with sanguinarine was measured by MTT assay. Apoptotic cells stained with Annexin V and 7-AAD were detected by flow cytometry. Mitochondrial membrane potential and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. The in vitro kinase activity of MELK was analyzed by using HTRF® KinEASE™-STK kit. The expression of proteins were determined using Western blotting and immunohistochemistry. Co-immunoprecipitation and immunofluorecence were used to study the interaction between STRAP and MELK. The anti-neoplastic effect of sanguinarine was observed in vivo in an orthotopic CRC model. RESULTS: Sanguinarine decreased the tumor size in a dose-dependent manner in orthotopical colorectal carcinomas through intrinsic apoptosis pathway in BALB/c-nu mice. It significantly increased cleavage of caspase 3 and PARP in implanted colorectal tissues. Sanguinarine increased mitochondrial ROS and triggered mitochondrial outer membrane permeabilization in multiple colorectal cancer (CRC) cell lines. NAC pretreatment lowered ROS level and downregulated apoptosis induced by sanguinarine. The intrinsic apoptosis induced by sanguinarine was Bax-dependent. The elevated expression and association between serine-threonine kinase receptor-associated protein (STRAP) and maternal embryonic leucine zipper kinase (MELK) were observed in Bax positive cells but not in Bax negative cells. Sanguinarine dephosphorylated STRAP and MELK and disrupted the association between them in HCT116 and SW480 cells. The expression and association between STRAP and MELK were also attenuated by sanguinarine in the tumor tissues. Importantly, we found that STRAP and MELK were overexpressed and highly phosphorylated in colorectal adenocarcinomas and their expression were significantly correlated with tumor stages. Furthermore, the expression of MELK, but not STRAP, was associated with lymph node metastasis. CONCLUSIONS: Sanguinarine dephosphorelates STRAP and MELK and disassociates the interaction between them to trigger intrinsic apoptosis. Overexpression of STRAP and MELK may be markers of CRC and their disassociation may be a determinant of therapeutic efficacy.
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Apoptose/efeitos dos fármacos , Benzofenantridinas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Isoquinolinas/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Benzofenantridinas/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Humanos , Isoquinolinas/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Papaveraceae/química , Permeabilidade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas de Ligação a RNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To test the hypothesis that modified Shenlingbaizhu decoction (MSD) attenuates the formation of intestinal adenomas by regulating activation of CD4+CD25+ forkhead box P3 (FoxP3) regulatory T cells (Tregs) by downregulation of hypoxia-inducible factor 1α (HIF-1α). METHODS: Chemical fingerprints of ginsenoside Rb1, ginsenoside Rc, paeoniflorin, and dioscin in standard extractions were used as material bases of MSD. Adenomatous polyposis coli multiple intestinal neoplasia (ApcMin/+) mice, which harbor a mutation in adenomatous polyposis coli, were used to host intestinal adenomas. Peripheral blood and spleen Tregs were analyzed by flow cytometry. Protein expression was analyzed by immunohistochemistry and Western blotting. RESULTS: The number and size of intestinal adenomas were significantly reduced by MSD treatment. Mucosal thickening and the spleen size were also substantially decreased by MSD. The carcinogenesis process in ApcMin/+ mice resembled that of human colorectal cancer. Molecular markers of neoplasms, such as ß-catenin, cyclooxygenase-2, proliferating cell nuclear antigen, and p53, were substantially ameliorated by MSD treatment. Moreover, MSD downregulated peripheral and spleen CD4+CD25+FoxP3+ Tregs and reduced in situ expression of CD4, CD25, and FoxP3 in intestinal adenomas. MSD also suppressed HIF-1α expression in the intestinal adenomas, and HIF-1α inhibition decreased expression of FoxP3 in Jurkat T cells under hypoxic conditions. CONCLUSION: MSD is a valid prescription to control the formation of intestinal adenomas in ApcMin/+ mice. It exerts anti-cancer effects partially through suppression of HIF-1α that induced activation of CD4+CD25+FoxP3+ Tregs in vivo and in vitro.
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OBJECTIVE: To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis. METHODS: MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting. RESULTS: Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells. CONCLUSION: Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.