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Arsenic trioxide inhibits the growth of cancer stem cells derived from small cell lung cancer by downregulating stem cell-maintenance factors and inducing apoptosis via the Hedgehog signaling blockade.

ABSTRACT: Background:Small cell lung cancer (SCLC) is the most deadly and aggressive type of primary lung cancer, with the 5-year survival rate lower than 5%. The FDA has approved arsenic trioxide (As2O3) for acute promyelocytic leukemia (APL) treatment. However, its role in SCLC-derived cancer stem cells (CSCs) remains largely unknown. Methods:CSCs were enriched from SCLC cell lines by culturing them as spheres in conditioned serum-free medium. Then, qPCR, western blot, serial passage, limiting dilution, Transwell, and tumorigenesis assay were performed to verify the cells' stem phenotypic characteristics. Anticancer efficiency of As2O3 was assessed in these cells using CCK8, colony formation, sphere formation, flow cytometry, qPCR, western blot analysis in vitro, and tumor growth curve, immunofluorescence, and TUNEL staining analyses in vivo. Results:The fifth-passage SCLC spheres showed a potent self-renewal capacity, higher clonal formation efficiency (CFE), SOX2, c-Myc, NANOG, and OCT4 levels, and invasion ability, and stronger tumorigenesis capacity than the parental SCLC cells, indicating that the SCLC sphere cells displayed CSC features. As2O3 inhibited the proliferation, clonality and sphere forming ability of SCLC-derived CSCs and suppressed the tumor growth of CSCs-derived xenograft tumors. As2O3 induced apoptosis and downregulation of SOX2 and c-Myc in vitro and in xenografts. Besides, SOX2 knockdown suppressed SCLC-derived CSCs to self-renew and induced apoptosis. Mechanistically, expression of GLI1 (a key transcription factor of Hedgehog pathway) and its downstream genes increased in SCLC-derived CSCs, compared to the parental cells. As2O3 dramatically downregulated GLI1 and its downstream genes in vitro and in vivo. The GLI inhibitor (GANT-61) recapitulated and enhanced the effects of As2O3 on SCLC-derived CSCs, including growth suppression, apoptosis induction, and GLI1, SOX2 and c-Myc downregulation. Conclusions:Altogether, As2O3 effectively suppressed SCLC-derived CSCs growth by downregulating stem cell-maintenance factors and inducing apoptosis. These effects are mediated at least partly via the Hedgehog signaling blockade.

SUBMITTER: Chang KJ

PROVIDER: S-EPMC7481635 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Arsenic trioxide inhibits the growth of cancer stem cells derived from small cell lung cancer by downregulating stem cell-maintenance factors and inducing apoptosis via the Hedgehog signaling blockade.

Chang Ke-Jie KJ Yin Ji-Zhong JZ Huang Hai H Li Bing B Yang Meng-Hang MH

Translational lung cancer research 20200801 4

<h4>Background</h4>Small cell lung cancer (SCLC) is the most deadly and aggressive type of primary lung cancer, with the 5-year survival rate lower than 5%. The FDA has approved arsenic trioxide (As<sub>2</sub>O<sub>3</sub>) for acute promyelocytic leukemia (APL) treatment. However, its role in SCLC-derived cancer stem cells (CSCs) remains largely unknown.<h4>Methods</h4>CSCs were enriched from SCLC cell lines by culturing them as spheres in conditioned serum-free medium. Then, qPCR, western blo ...[more]

PMID: 32953511

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Project description:Arsenic trioxide (ATO) is approved for treating terminal-stage liver cancer in China. Cryptotanshinone (CT), a STAT3 inhibitor, has exhibited certain anti-tumor potency; however, the use of CT enhanced ATO for treating liver cancer has not been reported. Here we try to elucidate how CT could enhance the efficacy of ATO for treating liver cancer and its correlation to STAT3 in vitro and in vivo.Cell viability of ATO combined with CT was assessed by 1MTT assay. Cell apoptosis induced by ATO combined with CT was detected by Annexin V/PI staining and apoptosis-related proteins were detected by western blotting. STAT3-related proteins were analysis by western blotting analysis and Immunofluorescence assays. Efficacy evaluation of ATO combined with CT on xenograft was carried in nude mice and related proteins were analysis by Immunohistochemistry assays.First we evaluated cell vitality, and our data indicated that the ATO combined with CT showed obvious growth inhibition of Bel-7404 cells compared to ATO or CT alone. Next we found that ATO combined with CT induced cell apoptosis in Bel-7404 cells and upregulated the activation of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-poly(ADP-ribose) polymerase in a time-dependent manner. Next, we found that ATO combined with CT not only inhibited the constitutive levels of phosphorylated-JAK2 and phosphorylated-STAT3Tyr705 but did so in a time-dependent manner. We also found that ATO combined with CT reversed the upregulated expression of phosphorylated-STAT3Tyr705 stimulated by interleukin-6 and downregulated STAT3 direct target genes and the anti-apoptotic proteins Bcl-2, XIAP, and survivin but obviously upregulated the promoting apoptosis proteins Bak,.In vivo studies showed that ATO combined with CT decreased tumor growth. Tumors from ATO combined with CT-treated mice showed decreased levels of phosphorylated-STAT3Tyr705 and the anti-apoptotic protein Bcl-2 but an increased level of pro-apoptotic protein Bax.Our study provides strong evidence that CT could enhance the efficacy of ATO in treating liver cancer both in vitro and in vivo. Downregulation of phosphorylated-STAT3 expression may play an important role in inducing apoptosis of Bel-7404 cells.

Project description:BackgroundMultiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by severe white matter demyelination. Because of its complex pathogenesis, there is no definite cure for MS. Experimental autoimmune encephalomyelitis (EAE) is an ideal animal model for the study of MS. Arsenic trioxide (ATO) is an ancient Chinese medicine used for its therapeutic properties with several autoimmune diseases. It is also used to inhibit acute immune rejection due to its anti-inflammatory and immunosuppressive properties. However, it is unclear whether ATO has a therapeutic effect on EAE, and the underlying mechanisms have not yet been clearly elucidated. In this study, we attempted to assess whether ATO could be used to ameliorate EAE in mice.MethodsATO (0.5 mg/kg/day) was administered intraperitoneally to EAE mice 10 days post-immunization for 8 days. On day 22 post-immunization, the spinal cord, spleen, and blood were collected to analyze demyelination, inflammation, microglia activation, and the proportion of CD4+ T cells. In vitro, for mechanistic studies, CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice and treated with ATO and then used for an apoptosis assay, JC-1 staining, imaging under a transmission electron microscope, and western blotting.ResultsATO delayed the onset of EAE and alleviated the severity of EAE in mice. Treatment with ATO also attenuated demyelination, alleviated inflammation, reduced microglia activation, and decreased the expression levels of IL-2, IFN-γ, IL-1β, IL-6, and TNF-α in EAE mice. Moreover, the number and proportion of CD4+ T cells in the spinal cord, spleen, and peripheral blood were reduced in ATO-treated EAE mice. Finally, ATO induced CD4+ T cell apoptosis via the mitochondrial pathway both in vitro and in vivo. Additionally, the administration of ATO had no adverse effect on the heart, liver, or kidney function, nor did it induce apoptosis in the spinal cord.ConclusionsOverall, our findings indicated that ATO plays a protective role in the initiation and progression of EAE and has the potential to be a novel drug in the treatment of MS.

Project description:Arsenic trioxide (ATO) has been used to treat acute promyelocytic leukemia. However, it is not effective in treating solid tumors such as colorectal cancer. We have previously reported that polyploid giant cancer cells (PGCCs) exhibiting the characteristics of cancer stem cells can be generated by various inducers. In this study, ATO was used to induce the formation of PGCCs in LoVo and Hct116 colon cancer cell lines. The migration, invasion, and proliferation abilities of colon cancer cells with and without ATO treatment were assessed by wound-healing, transwell, and plate colony formation assays. The expression of epithelial to mesenchymal transition-related proteins and erythroid differentiation-related proteins in colon cancer cells was further evaluated by western blot and immunocytochemical assays. LoVo and Hct116 cells were transfected with a eukaryotic expression vector for green fluorescent protein (GFP), red fluorescent protein (RFP), H2B-GFP, and H2B-mCherry to study PGCCs formation via cell fusion. WB and ICC assays were performed to assess the expression of cell fusion-related proteins. MG132, small interfering RNA-glial cell missing 1 (GCM1), and chromatin immunoprecipitation-polymerase chain reaction assays were performed to study the role of GCM1/syncytin-1-mediated cell fusion. Clinically, the significance of cell fusion-related proteins and erythroid differentiation-related proteins expression in human colorectal cancer tissues was evaluated. Results of our study showed that ATO induced the formation of PGCCs, and the daughter cells derived from PGCCs gained a mesenchymal phenotype and exhibited strong migration, invasion, and proliferation abilities. PGCCs also produced embryonic hemoglobin-delta and -zeta with strong oxygen-binding ability and erythroid differentiation-related proteins after ATO treatment. In addition, cell fusion was observed during the formation of PGCCs, indicated by the presence of yellow fluorescence via the GCM1/syncytin-1 signaling pathway. Clinically, the expression of cell fusion-related and erythroid differentiation-related proteins gradually increased with the progression of human colorectal cancer tissues. In conclusion, ATO can promote tumor progression by inducing the formation of PGCCs via GCM1/syncytin-1-mediated cell fusion. PGCCs can produce daughter cells with high invasion and migration abilities and embryonic hemoglobin with strong oxygen binding ability, promoting survival of tumor cells in a hypoxic microenvironment.

Project description:BackgroundThe evasion of apoptosis through dysregulated Bcl-2 family members is a hallmark of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). Therefore, targeting Bcl-2 with venetoclax has been suggested as an attractive strategy for inducing apoptosis in AML LSCs. However, the selective inhibition of Bcl-2 in AML often leads to upregulation of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance.MethodsWe assessed the combined effect of venetoclax and arsenic trioxide (ATO) on leukaemic cell viability, apoptosis, combination index, and cell cycle in the human LSC-like KG1 and KG1a cells. The synergistic effect of venetoclax and ATO on apoptosis was also examined in primary CD34+ and CD34+CD38- LSCs from the bone marrow (BM) of AML patients, and compared with those from healthy donors.ResultsVenetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with ATO; their synergism was aptly represented by the combination index. The combination of venetoclax and ATO impaired cell cycle progression by restricting cells within the sub-G1 phase and facilitating caspase-dependent apoptotic cell death associated with the loss of mitochondrial membrane potential, while sparing healthy BM haematopoietic stem cells. Mechanistically, ATO mitigated venetoclax-induced upregulation of Mcl-1 by the inhibition of AKT and ERK, along with activation of GSK-3β. This led to the Mcl-1 destabilisation, triggering Noxa and Bim to facilitate apoptosis and the consequent activation of the apoptosis executioner protein Bak. Moreover, the combination promoted phosphorylation of ATM, Chk2, p38, and H2AX, indicating an active DNA damage response.ConclusionsOur findings demonstrate the synergistic, preferential antileukaemic effects of venetoclax and ATO on LSCs, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat acute leukaemia.

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Arsenic trioxide inhibits the growth of cancer stem cells derived from small cell lung cancer by downregulating stem cell-maintenance factors and inducing apoptosis via the Hedgehog signaling blockade.

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Arsenic trioxide inhibits the growth of cancer stem cells derived from small cell lung cancer by downregulating stem cell-maintenance factors and inducing apoptosis via the Hedgehog signaling blockade.

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