Shikonin suppresses proliferation and induces apoptosis in endometrioid endometrial cancer cells via modulating miR-106b/PTEN/AKT/mTOR signaling pathway

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, which exerts anticancer effects in various cancers. However, the molecular mechanisms underlying the therapeutic effects of shikonin against endometrioid endometrial cancer (EEC) have not yet been fully elucidated. Herein, we investigated anticancer effects of shikonin on EEC cells and explored the underlying molecular mechanism. We observed that shikonin inhibits proliferation in human EEC cell lines in a dose-dependent manner. Moreover, shikonin-induced apoptosis was characterized by the up-regulation of the pro-apoptotic proteins cleaved-Caspase-3 and Bax, and the down-regulation of the anti-apoptotic protein Bcl-2. Microarray analyses demonstrated that shikonin induces many miRNAs’ dysregulation, and miR-106b was one of the miRNAs being most significantly down-regulated. miR-106b was identified to exert procancer effect in various cancers, but in EEC remains unclear. We first confirmed that miR-106b is up-regulated in EEC tissues and cells, and knockdown of miR-106b suppresses proliferation and promotes apoptosis. Meanwhile, our results validated that the restored expression of miR-106b abrogates the antiproliferative and pro-apoptotic effects of shikonin. We also identified that miR-106b targets phosphatase and tensin homolog (PTEN), a tumor suppressor gene, which in turn modulates AKT/mTOR signaling pathway. Our findings indicated that shikonin inhibits proliferation and promotes apoptosis in human EEC cells by modulating the miR-106b/PTEN/AKT/mTOR signaling pathway, suggesting shikonin could act a potential therapeutic agent in the EEC treatment.

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Ginsenoside Rg3 Suppresses Proliferation and Induces Apoptosis in Human Osteosarcoma

BioMed Research International ◽

10.1155/2018/4306579 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Yi Li ◽

Jinying Lu ◽

Furong Bai ◽

Yanan Xiao ◽

Yiran Guo ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathway ◽

Mtor Signaling ◽

Assay Method ◽

Osteosarcoma Cell ◽

Dependent Manner ◽

Ginsenoside Rg3 ◽

Mtor Signaling Pathway ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells

Osteosarcoma is the most common primary malignancy of bone in children and the elderly. Recently, more and more researches have demonstrated that Ginsenoside Rg3 (Rg3) is involved in chemotherapy resistance in many cancer, making it a promising Chinese herbal monomer for oncotherapy. In this study, we investigated the efficacy of Rg3 in human osteosarcoma cell lines (MG-63, U-2OS, and SaOS-2). Cell proliferation was measured by CCK8 assay. The migration of cells was examined using the scratch assay method. Quantification of apoptosis was assessed further by flow cytometry. In addition, the expression of apoptosis-related genes (caspase9, caspase3, Bcl2, and Bax) were investigated using RT-PCR. We further investigated the protein level expression of Bcl 2, cleaved-caspase3, and PI3K/AKT/mTOR signaling pathway factors by Western blot assay. Our results revealed that Rg3 inhibited the proliferation and migration of human osteosarcoma cells and induced apoptosis in a concentration- and time-dependent manner. Western blot results showed that Rg3 reduced the protein expression of Bcl2 and PI3K/AKT/mTORbut increased the levels of cleaved-caspase3. Therefore, we hypothesized Rg3 inhibits the proliferation of osteosarcoma cell line and induces their apoptosis by affecting apoptosis-related genes (Bcl2, caspase3) as well as the PI3K/AKT/mTOR signaling pathway. To conclude, Rg3 is a new therapeutic agent against osteosarcoma.

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Melatonin-induced Cell Rejuvenation from Long-term Ex-vivo passaging Functioned by its Restoration of Damaged Cell Autophagic Processes

10.21203/rs.3.rs-122498/v1 ◽

2020 ◽

Author(s):

Yi-Zhou Tan ◽

Xin-Yue Xu ◽

Ji-Min Dai ◽

Yuan Yin ◽

Xiao-Tao He ◽

...

Keyword(s):

Stem Cells ◽

Signaling Pathway ◽

Cellular Senescence ◽

Ex Vivo ◽

Mtor Signaling ◽

Beclin 1 ◽

Dependent Manner ◽

Mtor Signaling Pathway ◽

Associated Proteins

Abstract Background: Stem cells undergone long-term ex-vivo expansion are most likely functionally compromised (namely cellular senescence) in terms of their stem cell properties and therapeutic potentials. Due to the ability to attenuate cellular senescence, melatonin (MLT) has been proposed as an adjuvant across long-term cell expansion protocols, but the underlying mechanism remains largely unknown. Methods: Human periodontal ligament stem cells (PDLSCs) were isolated and cultured ex-vivo for 15 passages, and passage 2, 7 and 15 cells were used to interrogate the cellular senescence and alteration in cell autophagy during long-term expansion. The cellular senescence features were evidenced by senescence-associated β-galacotosidase (SA-β-gal) activity and the expression of senescence-related proteins including p53, p21, p16 and γ-H2AX. Electronic microscope was used to observe the autophagic vesicles. Adenovirus mRFP-GFP-LC3 was transfected to indicate the alteration of autophagic flux during long-term expansion, and the autophagy-associated proteins Atg7, Beclin-1, LC3-II and p62 were evaluated by Western blot. Results: It was found that long-term in-vitro passaging led to an accumulated SA-β-gal, elevated expressions of p53, p21, p16 and γ-H2AX, along with downregulated autophagy-associated proteins Atg7, Beclin-1 and LC3 as well as a mounting autophagy substrate p62. In accordance with expectation, supplemented with MLT not only ameliorated cells to a younger state but also restored the impaired autophagy level in senescent cells. Additionally, we demonstrated that autophagy inhibitor could block such MLT-induced cell rejuvenation. When the underlying signaling pathways involved was interrogated, we found that MLT receptor (MT) participated in mediating MLT-related autophagy restoration by regulating PI3K/AKT/mTOR signaling pathway.Conclusions: The present study suggests that MLT may rejuvenate long-term expansion-caused cellular senescence by restoring autophagy, more likely via PI3K/AKT/mTOR signaling pathway in an MT-dependent manner. This is the first report identifying the MT-dependent PI3K/AKT/mTOR signaling involved in MLT-induced autophagy alteration, pointing to a potential target for using autophagy-restoring agents such as MLT to develop optimized clinical-scale cell production protocols.

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Activated mTORC1-SIRT6 Mediates Myofibroblast Differentiation and Idiopathic Pulmonary Fibrosis

10.21203/rs.3.rs-587874/v1 ◽

2021 ◽

Author(s):

Ji Zhang ◽

Yi Hu ◽

Huiping Huang ◽

Qun Liu ◽

Yang Du ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Signaling Pathway ◽

Mtor Signaling ◽

Signalling Pathway ◽

Lung Fibroblasts ◽

Myofibroblast Differentiation ◽

Dependent Manner ◽

Mtor Signaling Pathway ◽

Mtorc1 Pathway ◽

Tgf Β1

Abstract BackgroundIdiopathic pulmonary fibrosis (IPF) is characterised by accumulation of myofibroblasts and deposition of extracellular matrix proteins. Fibroblast-to-myofibroblast transdifferentiation and myofibroblast hyperproliferation plays a major role in pulmonary fibrosis. Moreover, mTOR signaling pathway and SIRT6 play a critical role in pulmonary fibrosis. However, the mechanisms whether SIRT6 affect the myofibroblasts differentiation during IPF remain unclear.MethodWe investigated myofibroblast differentiation using a bleomycin-induced mouse pulmonary fibrosis model and TGF-b1 induced human fetal lung fibroblasts (MRC5) in vitro. We used both SIRT6 siRNA and rapamycin to study the role of SIRT6 and mTOR signaling pathway in the normal human lung fibroblasts and the myofibroblasts from human IPF lungs.ResultsOur data show that high level of SIRT6 was detected in IPF samples, and SIRT6 was signiﬁcantly upregulated by TGF-β1 in a time and concentration-dependent manner. SIRT6 expression and activation of mTORC1 signalling pathway were upregulated in fibrotic lung tissues and primary lung fibroblasts isolated from patients with IPF and bleomycin-challenged mice. Furthermore, rapamycin treatment inhibited mTORC1 pathway activity and SIRT6 protein expression. SIRT6 SiRNA failed to mediate the activity of mTORC1 pathway and autophagy induction. However, SIRT6 knockdown could promote TGF-b1 induced pro-fibrotic cytokines.ConclusionActivated mTORC1 signalling pathway regulated SIRT6 overexpression. Deficiency of SIRT6 mediated myofibroblasts differentiation through induced pro-fibrotic cytokines production in the present of TGF-β1. The study indicated that manipulations of SIRT6 expression may provide a new therapeutic strategy to prevent and reverse the progression of pulmonary fibrosis.

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mTOR Signaling Pathway Regulates Sperm Quality in Older Men

Cells ◽

10.3390/cells8060629 ◽

2019 ◽

Vol 8 (6) ◽

pp. 629 ◽

Cited By ~ 3

Author(s):

Silva ◽

Cabral ◽

Correia ◽

Carvalho ◽

Sousa ◽

...

Keyword(s):

Signaling Pathway ◽

Molecular Mechanisms ◽

Sperm Quality ◽

Older Men ◽

Mtor Signaling ◽

Paternal Age ◽

Signaling Proteins ◽

Mtor Signaling Pathway ◽

Mtorc1 Signaling ◽

The Impact

: Understanding how age affects fertility becomes increasingly relevant as couples delay childbearing toward later stages of their lives. While the influence of maternal age on fertility is well established, the impact of paternal age is poorly characterized. Thus, this study aimed to understand the molecular mechanisms responsible for age-dependent decline in spermatozoa quality. To attain it, we evaluated the impact of male age on the activity of signaling proteins in two distinct spermatozoa populations: total spermatozoa fraction and highly motile/viable fraction. In older men, we observed an inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) in the highly viable spermatozoa population. On the contrary, when considering the entire spermatozoa population (including defective/immotile/apoptotic cells) our findings support an active mTORC1 signaling pathway in older men. Additionally, total spermatozoa fractions of older men presented increased levels of apoptotic/stress markers (e.g., cellular tumor antigen p53-TP53) and mitogen-activated protein kinases (MAPKs) activity. Moreover, we established that the levels of most signaling proteins analyzed were consistently and significantly altered in men more than 27 years of age. This study was the first to associate the mTOR signaling pathway with the age impact on spermatozoa quality. Additionally, we constructed a network of the sperm proteins associated with male aging, identifying TP53 as a central player in spermatozoa aging.

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Assessing the efficacy of targeting the phosphatidylinositol 3-kinase/AKT/mTOR signaling pathway in endometrial cancer

Gynecologic Oncology ◽

10.1016/j.ygyno.2014.02.022 ◽

2014 ◽

Vol 133 (2) ◽

pp. 346-352 ◽

Cited By ~ 26

Author(s):

Leslie S. Bradford ◽

Alejandro Rauh-Hain ◽

Rachel M. Clark ◽

Jolijn W. Groeneweg ◽

Ling Zhang ◽

...

Keyword(s):

Endometrial Cancer ◽

Signaling Pathway ◽

Mtor Signaling ◽

Mtor Signaling Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Estrogen promotes fat mass and obesity-associated protein nuclear localization and enhances endometrial cancer cell proliferation via the mTOR signaling pathway

Oncology Reports ◽

10.3892/or.2016.4613 ◽

2016 ◽

Vol 35 (4) ◽

pp. 2391-2397 ◽

Cited By ~ 14

Author(s):

YAPING ZHU ◽

JIAQI SHEN ◽

LIYAN GAO ◽

YOUJI FENG

Keyword(s):

Cell Proliferation ◽

Endometrial Cancer ◽

Signaling Pathway ◽

Cancer Cell ◽

Nuclear Localization ◽

Fat Mass ◽

Mtor Signaling ◽

Cancer Cell Proliferation ◽

Mtor Signaling Pathway ◽

Endometrial Cancer Cell

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AMP-Activated Protein Kinase Alleviates Extracellular Matrix Accumulation in High Glucose-Induced Renal Fibroblasts through mTOR Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000369687 ◽

2015 ◽

Vol 35 (1) ◽

pp. 191-200 ◽

Cited By ~ 15

Author(s):

Xia Luo ◽

Lingyan Deng ◽

Laxmi Pangeni Lamsal ◽

Wenjuan Xu ◽

Cheng Xiang ◽

...

Keyword(s):

Extracellular Matrix ◽

Protein Kinase ◽

Signaling Pathway ◽

High Glucose ◽

Mtor Signaling ◽

Dependent Manner ◽

Mtor Signaling Pathway ◽

Matrix Synthesis ◽

Matrix Accumulation ◽

Amp Activated Protein Kinase

Background/Aims: Extracellular matrix accumulation contributes significantly to the pathogenesis of diabetic nephropathy. Although AMP-activated protein kinase (AMPK) has been found to inhibit extracellular matrix synthesis by experiments in vivo and vitro, its role in alleviating the deposition of extracellular matrix in renal interstitial fibroblasts has not been well defined. Methods: Currently, we conducted this study to investigate the effects of AMPK on high glucose-induced extracellular matrix synthesis and involved intracellular signaling pathway by using western blot in the kidney fibroblast cell line (NRK-49f). Results: Collagen IV protein levels were significantly increased by high glucose in a time-dependent manner. This was associated with a decrease in Thr72 phosphorylation of AMPK and an increase in phosphorylation of mTOR on Ser2448. High glucose-induced extracellular matrix accumulation and mTOR activation were significantly inhibited by the co-treatment of rAAV-AMPKα1312 (encoding constitutively active AMPKα1) whereas activated by r-AAV-AMPKα1D157A (encoding dominant negative AMPKα1). In cultured renal fibroblasts, overexpression of AMPKα1D157A upregulated mTOR signaling and matrix synthesis, which were ameliorated by co-treatment with the inhibitor of mTOR, rapamycin. Conclusion: Collectively, these findings indicate that AMPK exerts renoprotective effects by inhibiting the accumulation of extracellular matrix through mTOR signaling pathway.

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Aescin Protects Neuron from Ischemia-Reperfusion Injury via Regulating the PRAS40/mTOR Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/7815325 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Xinjie Gao ◽

Heng Yang ◽

Jiabin Su ◽

Weiping Xiao ◽

Wei Ni ◽

...

Keyword(s):

Reperfusion Injury ◽

Signaling Pathway ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Intravenous Thrombolysis ◽

Mtor Signaling ◽

Dependent Manner ◽

Protective Effects ◽

Mtor Signaling Pathway

Ischemic stroke is one of the major causes of disability; widely use of endovascular thrombectomy or intravenous thrombolysis leads to more attention on ischemia-reperfusion injury (I/R injury). Aescin, a natural compound isolated from the seed of the horse chestnut, has been demonstrated anti-inflammatory and antiedematous effects previously. This study was aimed at determining whether aescin could induce protective effects against ischemia-reperfusion injury and exploring the underlying mechanisms in vitro. Primary cultured neurons were subjected to 2 hours of oxygen-glucose deprivation (OGD) followed by 24 hours of simulated reperfusion. Aescin, which worked in a dose-dependent manner, could significantly attenuate neuronal death and reduce lactate dehydrogenase (LDH) release after OGD and simulated reperfusion. Aescin treatment at a concentration of 50 μg/ml provided protection with fewer side effects. Results showed that aescin upregulated the phosphorylation level of PRAS40 and proteins in the mTOR signaling pathway, including S6K and 4E-BP1. However, PRAS40 knockdown or rapamycin treatment was able to undermine and even abolish the protective effects of aescin; meanwhile, the levels of phosphorylation PRAS40 and proteins in the mTOR signaling pathway were obviously decreased. Hence, our study demonstrated that aescin provided neuronal protective effects against I/R injury through the PRAS40/mTOR signaling pathway in vitro. These results might contribute to the potential clinical application of aescin and provide a therapeutic target on subsequent cerebral I/R injury.

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DHOK Exerts Anti-Cancer Effect Through Autophagy Inhibition in Colorectal Cancer

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.760022 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuhan Shu ◽

Xin Sun ◽

Guiqin Ye ◽

Mengting Xu ◽

Zhipan Wu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

Mammalian Target Of Rapamycin ◽

Mtor Signaling ◽

Dependent Manner ◽

Mtor Signaling Pathway ◽

Eurycoma Longifolia ◽

Anti Cancer ◽

Lc3 Puncta

DHOK (14,15β-dihydroxyklaineanone) is a novel diterpene isolated from roots of Eurycoma longifolia Jack, a traditional herb widely applied in Southeast Asia. It is reported that DHOK has cytotoxic effect on cancer cells, but its anti-cancer mechanism has still been not clear. In our study, we first observed that DHOK inhibits cell proliferation of colorectal cancer cells in a time- and dose-dependent manner. Next, we performed transcriptome sequencing to identify the targets of DHOK and found that autophagy-related signaling pathways are involved under DHOK treatment. Indeed, in DHOK-treated cells, the level of autophagosome marker LC3 and the formation of GFP-LC3 puncta were decreased, indicating the reduction of autophagy. Moreover, confocal microscopy results revealed the lysosomal activity and the formation of autolysosomes are also inhibited. Our western blotting results demonstrated the activation of mammalian target of rapamycin (mTOR) signaling pathway by DHOK, which may be attributed to the enhancement of ERK and AKT activity. Functionally, activation of autophagy attenuated DHOK-caused cell death, indicating that autophagy serves as cell survival. In xenograft mouse model, our results also showed that DHOK activates the mTOR signaling pathway, decreases autophagy level and inhibits the tumorigenesis of colon cancer. Taken together, we revealed the molecular mechanism of DHOK against cancer and our results also demonstrate great potential of DHOK in the treatment of colorectal cancer.

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PRDX2 Performs the Oncogenic Functions in Ewing’s Sarcoma

10.21203/rs.3.rs-51646/v1 ◽

2020 ◽

Author(s):

Ruifeng Xue ◽

Zhengfu Fan ◽

Yunhe An

Keyword(s):

Western Blot ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Ewing’S Sarcoma ◽

Ewing's Sarcoma ◽

Es Cells ◽

Mtor Signaling ◽

Mtor Signaling Pathway ◽

Strong Basis ◽

Apoptotic Protein

Abstract Background: Ewing's sarcoma (ES) is the second most common malignant primary bone tumor in children and adolescents, characterized by malignant proliferation of small round cells. The survival rate of this disease continues to be low. Peroxiredoxin2 (PRDX2) is a multifunctional peroxidase family member with anti-oxidation, involvement in intracellular signaling, chaperones, and tumor. But the function and underlying mechanism of PRDX2 in ES is still unknown. Herein we investigated the role and mechanism of PRDX2 in the development of ES, and tested its potential for the treatment of ES.Methods: We explored the function of PRDX2 on ES through knocking down the expression of NKAP in A673 and RDES cells by siRNA interference (siPRDX2). We examined the effects of siPRDX2 on cell motility and apoptosis using CCK8, colony formation, transwell, gelatin zymography, flow cytometry, PI/Hoechst33342 double dye and western blot assays. In addition, western blot was used to analyze the activation of the AKT/mTOR signaling pathway.Results: Here we showed that downregulation of PRDX2 strongly inhibited the motility of A673 and RDES cells. Interestingly, siPRDX2 induced cell apoptosis. Furthermore, the expression of anti-apoptotic protein Bcl2 in siPRDX2 group was significantly decreased, while the expression of pro-apoptotic protein Bax and cleaved Caspase-9 was strongly increased. Finally to identify the molecular mechanisms involved, we examined related proteins of the AKT/mTOR signaling pathway and found that siPRDX2 significantly inhibited the phosphorylation of AKT and the expression of Cyclin D1.Conclusion: These observations suggest that siPRDX2 inhibited the ES cells motility and induced apoptosis in which the AKT/mTOR signaling pathway involved. The enhanced understanding to this molecular mechanism has provided a strong basis for the development of novel therapeutic strategies for ES.

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