Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity

Kamel, Walied; Sugihara, Eiji; Nobusue, Hiroyuki; Yamaguchi-Iwai, Sayaka; Onishi, Nobuyuki; Maki, Kenta; Fukuchi, Yumi; Matsuo, Koichi; Muto, Akihiro; Saya, Hideyuki; Shimizu, Takatsune

doi:10.1158/1535-7163.mct-16-0499

Cited by 75 publications

(82 citation statements)

References 40 publications

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“…To identify effective therapeutic drugs for osteosarcoma, we screened 1164 US Food and Drug Administration (FDA)–approved compounds all at the same concentration (1.92 μM), using a novel humanoid robot (Robotic Biology Institute, Tokyo, Japan) . Calcitriol decreased the viability of AXT cells by 44% of the control level (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…In a previous work, we generated a mouse model of osteosarcoma by overexpressing c‐MYC in bone marrow stromal cells derived from Ink4a/Arf knockout mice . Inoculation of these cells, designated AXT cells, into syngeneic C57BL/6 mice results in rapid formation of lethal tumors with metastatic lesions that faithfully mimic human osteoblastic osteosarcoma, providing a useful model for preclinical studies …”

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confidence: 99%

“…We screened >1100 FDA‐approved drugs in AXT cells in vitro, and identified calcitriol as a drug candidate with therapeutic potential against osteosarcoma. Calcitriol, [1,25(OH) 2 D 3 ], the most biologically active hormonal form of vitamin D, is synthesized from 25(OH)D 3 in the kidneys by the cytochrome P450 enzyme CYP27B1 .…”

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confidence: 99%

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Calcitriol exerts an anti‐tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

et al. 2017

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Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)‐approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti‐proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti‐osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.

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Section: Resultsmentioning

confidence: 99%

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Calcitriol exerts an anti‐tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

et al. 2017

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“…Simvastatin is therefore partially effective by inducing oxidative stress. Similar to the triggring effect of simvastatin on apoptosis of osteosarcoma cells [135], the stimulation of eryptosis by simvastatin on cell membrane scrambling requires apparently activity of p38 kinase, which is known to participate in the orchestration of eryptosis [53]. Accordingly, the effect of simvastatin on annexin-V-binding is significantly blunted in the presence of p38 kinase inhibitor SB203580.…”

Section: Discussionmentioning

confidence: 99%

Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

Bhuyan

Nüßle

Cao

et al. 2017

Cell Physiol Biochem

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Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺ and by addition of SB203580 (2 µM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca²⁺ entry, oxidative stress, and p38 kinase.

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“…Anticancer effects of statins have been reported in a variety of cancer types including osteosarcoma [29][30][31]. Although RhoAmediated signaling has been shown to be involved in simvastatin-induced apoptosis and atorvastatin-reduced invasion of osteosarcoma cells [17,32], Olivia et al identified CYR61 as a new target of atorvastatin that was independent from RhoA signaling [13]. CYR61 has been reported to be an oncogene and is associated with osteosarcoma aggressiveness and metastatic potential [11].…”

Section: Discussionmentioning

confidence: 99%

miR-33a Mediates the Anti-Tumor Effect of Lovastatin in Osteosarcoma by Targeting CYR61

Huang

Zhang

Shao

et al. 2018

Cell Physiol Biochem

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Background/Aims: Preventing cell metastasis is an effective therapeutic strategy to treat osteosarcoma and improve prognosis. Statins have been found to have anticancer effects in addition to their cholesterol-lowering action. As a new target of statins, cysteine-rich 61 (CYR61) was recently identified to promote cell migration and metastasis in osteosarcoma. However, the underlying mechanisms mediating the regulation of CYR61 expression by statins remain unknown. Methods: Human osteosarcoma cell lines MG63 and SaOS2 were used to clarify the effect of lovastatin on CYR61 expression. Real-time PCR was performed to detect mRNA or microRNA (miRNA) levels and western blot was performed to detect protein levels. Cell invasive ability was determined using Transwell assays. Lentivirus encoding CYR61 cDNA or sterol regulatory element-binding protein 2 (SREBP-2) shRNA was used to upregulate CYR61 expression or downregulate SREBP-2 expression. Binding of the CYR61 3’ untranslated region (UTR) and miR-33a was analyzed by luciferase reporter assay. Results: We found that lovastatin treatment decreased CYR61 expression, inhibited cell invasion and altered epithelial-to-mesenchymal-transition (EMT)-related protein expression, while CYR61 overexpression abolished the effect of lovastatin. Moreover, lovastatin increased the expression of SREBP-2 and miR-33a, which were then downregulated by SREBP-2 silencing. Bioinformatics analysis indicated that the CYR61 3′UTR harbored a potential miR-33a binding site and luciferase reporter assay demonstrated that CYR61 was a target of miR-33a in osteosarcoma cells. Furthermore, miR-33a could inhibit cell invasion and alter EMT-related protein expression. SREBP-2 silencing or miR-33a inhibitor upregulated CYR61 expression and reversed the effects of lovastatin on cell invasion and EMT-related proteins. Conclusion: Our findings suggest lovastatin suppresses osteosarcoma cell invasion through the SREBP-2/miR-33a/CYR61 pathway.

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Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity

Cited by 75 publications

References 40 publications

Calcitriol exerts an anti‐tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

Calcitriol exerts an anti‐tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

Simvastatin, a Novel Stimulator of Eryptosis, the Suicidal Erythrocyte Death

miR-33a Mediates the Anti-Tumor Effect of Lovastatin in Osteosarcoma by Targeting CYR61

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