Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here.HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation.Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor.MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues.ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors.ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.     

Loss of FAM60A attenuates cell proliferation in glioma via suppression of PI3K/Akt/mTOR signaling pathways

BackgroundGlioma is a common malignant tumor of the central nervous system with a high incidence and mortality. Family with sequence similarity 60 member A (FAM60A) is a new subunit of the Sin3 deacetylase complex. The clinical significance and biologic role of FAM60A in glioma remain unclear.MethodsThe expression of FAM60A in normal glial cells, glioma cells, and five-paired gliomas, and adjacent noncancerous tissues was quantified using real-time polymerase chain reaction (PCR) and western blotting. FAM60A protein expression in 179 archived, paraffin-embedded glioma samples was analyzed using immunohistochemistry. The roles of FAM60A in glioma cell proliferation and tumorigenicity were explored in vitro and in vivo. The underlying molecular mechanisms were elucidated using Western blot assay. Serum exosomal FAM60A levels of glioma patients were detected using electron microscopy, western blot, and real-time PCR.ResultsFAM60A expression was significantly up-regulated in glioma tissues and cell lines and positively associated with a worse outcome in glioma. Knockdown of FAM60A could inhibit glioma cell proliferation and tumorigenicity in vitro and in vivo. Besides, FAM60A expression was detectable in extracted serum exosomes with a higher expression in the glioma cancer group than in the normal group.ConclusionsLoss of FAM60A attenuates cell proliferation in glioma by suppressing PI3K/Akt/mTOR signaling pathways. Therefore, FAM60A may act as a prognostic biomarker and therapeutic target for glioma.     

SETD2 epidermal deficiency promotes cutaneous wound healing via activation of AKT/mTOR Signalling

ObjectivesCutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain–containing 2 (SETD2) is the only known histone H3K36 tri‐methylase; however, its role in skin wound healing remains unclear.Materials and MethodsTo elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis‐specific Setd2‐deficient mice. Wound‐healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound‐healing assays were performed on Setd2‐knockdown and Setd2‐overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA‐seq and H3K36me3 ChIP‐seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin).ResultsEpidermis‐specific Setd2‐deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure.ConclusionsOur results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling.     

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

Purpose: Carrimycin is a newly synthesized macrolide antibiotic with good antibacterial effect. Exploratory experiments found its function in regulating cell physiology, proliferation and immunity, suggesting its potential anti-tumor capacity. The aim of this study is to investigate the anti-tumor effect of carrimycin against human oral squamous cell carcinoma cells in vitro and in vivo.Methods: Human oral squamous cell carcinoma cells (HN30/HN6/Cal27/HB96 cell lines) were treated with gradient concentration of carrimycin. Cell proliferation, colony formation and migration ability were analyzed. Cell cycle and apoptosis were assessed by flow cytometry. The effect of carrimycin on OSCC in vivo was investigated in tumor xenograft models. Immunohistochemistry, western blot assay and TUNEL assays of tissue samples from xenografts were performed. The key proteins in PI3K/AKT/mTOR pathway and MAPK pathway were examined by western blot.Results: As the concentration of carrimycin increased, the proliferation, colony formation and migration ability of OSCC cells were inhibited. After treating with carrimycin, cell cycle was arrested in G0/G1 phase and cell apoptosis was promoted. The tumor growth of xenografts was significantly suppressed. Furthermore, the expression of p-PI3K, p-AKT, p-mTOR, p-S6K, p-4EBP1, p-ERK and p-p38 were down-regulated in vitro and in vivo.Conclusions: Carrimycin can inhibit the biological activities of OSCC cells in vitro and in vivo, and regulate the PI3K/AKT/mTOR and MAPK pathways.     

AKT Inhibitors Promote Cell Death in Cervical Cancer through Disruption of mTOR Signaling and Glucose Uptake

Background

PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability.

Experimental Design

Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with ¹⁸F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay.

Results

Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment.

Conclusions

The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.     

The Effect and Mechanism of LINC00663 on the Biological Behavior of Glioma

Glioma is the most frequent primary malignant brain tumor, which is characterized by high incidence and mortality, with a poor prognosis. Numerous studies have revealed the abnormal expression of long non-coding RNAs in gliomas. This study explored the effects and potential mechanism of LINC00663 in glioma. The LINC00663 levels and their prognostic values were analyzed from the GEO databases using bioinformatics. Also, LINC00663 expression in tissue samples and cell lines was measured using qRT-PCR. The roles of LINC00663 in glioma were confirmed using CCK8, EdU assay as well as Transwell tests. Moreover, the influences of LINC00663 on the AKT/mTOR signal cascades were detected using western blotting assay. LINC00663 expression was higher in both glioma tissues and cell lines than that in the normal brain tissues and human astrocytes. High expression of LINC00663 led to the low overall survival rate of patients with glioma. LINC00663 knockdown notably restrained cell proliferation, migration, and invasion abilities by decreasing the activation of AKT and mTOR. This study indicated that LINC00663 might have a cancer-promoting role in accelerating glioma development and progression through regulating AKT/mTOR pathway.

     

Quercetin Inhibits Angiogenesis Mediated Human Prostate Tumor Growth by Targeting VEGFR- 2 Regulated AKT/mTOR/P70S6K Signaling Pathways

Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.     

LncRNA MYU对胶质瘤细胞周期分布、增殖、转移和凋亡以及PI3K/Akt信号通路的影响

摘要 目的：探讨长链非编码RNA（LncRNA）MYU对胶质瘤细胞周期分布、细胞增殖、迁移、侵袭和凋亡的影响,并初步探讨其作用机制。方法：实时荧光定量PCR（RT-qPCR）检测人脑正常胶质细胞HEB和胶质瘤细胞（U-251MG、A172、SHG139）中LncRNA MYU的表达情况。选取SHG139细胞,分为正常对照（NC）组、si-con组、si-LncRNA MYU组进行转染实验,行RT-qPCR检测转染效果。分别采用流式细胞术、细胞计数试剂盒（CCK-8）、Transwell实验检测沉默LncRNA MYU对SHG139细胞周期分布和凋亡、细胞增殖、细胞迁移和侵袭的影响。蛋白免疫印迹（Western blot）法检测基质金属蛋白酶2（MMP-2）、MMP-9、裂解的半胱氨酸天冬氨酸蛋白酶3（Cleaved caspase-3）、Cleaved caspase-9以及磷脂酰肌醇-3-羟激酶/蛋白激酶B（PI3K/Akt）信号通路相关蛋白表达情况。结果：LncRNA MYU在胶质瘤细胞株中比人脑正常胶质细胞中的表达水平显著升高（P＜0.05）,因此选择表达量最高的SHG139细胞进行转染实验。沉默LncRNA MYU能够显著诱导SHG139细胞G0-G1期阻滞、抑制细胞增殖、迁移和侵袭并诱导细胞凋亡（P＜0.05）。沉默LncRNA MYU可显著抑制MMP-2、MMP-9、p-PI3K和p-AKT表达并促进Cleaved caspase-3、Cleaved caspase-9表达（P＜0.05）。结论：沉默LncRNA MYU可诱导胶质瘤细胞G0-G1期阻滞,抑制细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与抑制PI3K/AKT信号通路有关。     

Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.     

LncRNA DLGAP1-AS2 promotes the radioresistance of rectal cancer stem cells by upregulating CD151 expression via E2F1

BackgroundRadiotherapy resistance is one of the major causes of rectal cancer treatment failure. LncRNA DLGAP1-AS2 participates in the progression of several cancers. We explored the role and potential mechanism of DLGAP1-AS2 in the radioresistance of rectal cancer stem cells.MethodsHR8348-R cells, radioresistant cells from HR8348 after irradiation, were isolated into CD133 negative (CD133⁻) and positive (CD133⁺) cells. Cell proliferation, apoptosis, migration and tumorsphere formation were determined by CCK-8, flow cytometry, wound healing assay and tumorsphere formation assay, respectively. CD133, tumor stem cell drug resistance gene (MDR1 and BCRP1), DNA repair marker (γ-H2AX) and AKT/mTOR/cyclinD1 signaling were measured by Western blot. The relationship between DLGAP1-AS2 and E2F1 was verified using RIP. The interaction between E2F1 and CD151 promoter was confirmed using dual-luciferase reporter gene assay and ChIP. AKT inhibitor API-2 was employed for validating the effect of AKT/mTOR/cyclinD1 signaling in the radioresistance of rectal cancer cells.ResultsThe DLGAP1-AS2 level was increased in CD133⁺ cells after irradiation. DLGAP1-AS2 knockdown inhibited the proliferation, migration and tumorsphere formation while stimulating apoptosis in CD133⁺ cells. DLGAP1-AS2 inhibition downregulated the expression of CD133, MDR1, BCRP1 and γ-H2AX and suppressed AKT/mTOR/cyclinD1 activation. DLGAP1-AS2 upregulated the expression of CD151 by interacting with E2F1. API-2 neutralized the promotive effects of overexpressed CD151 on radioresistance.ConclusionDLGAP1-AS2 accelerates the radioresistance of rectal cancer cells through interactions with E2F1 to upregulate CD151 expression via the activation of the AKT/mTOR/cyclinD1 pathway.     

MK2206 Enhances Cisplatin-Induced Cytotoxicity and Apoptosis in Testicular Cancer Through Akt Signaling Pathway Inhibition

OBJECTIVE: To improve conventional chemotherapeutic efficacy, it is significant to identify novel molecular markers for chemosensitivity as well as possible molecules accelerating cell-killing mechanisms. In this study, we attempted to elucidate how MK2206, an allosteric Akt inhibitor, enhances the cisplatin (CDDP)-induced cytotoxicity and apoptosis in testicular cancer. MATERIALS AND METHODS: We checked three testicular cancer cell lines for the expression of phospho(p)-Akt and its downstream molecules targets by Western blot. The potential antitumor effects were analyzed by MTT assay in vitro and by subcutaneous xenograft models in vivo. The cell invasion was analyzed by transwell invasion assay, and the activities of Akt signaling pathway and expression of apoptosis-related proteins were measured by Western blot. RESULTS: Our results indicated that there was overactivation of p-Akt and its downstream molecules in testicular cancer cell lines compared with normal testis epithelium cells. MK2206 (600 nM) inhibited cell invasion in TCAM-2 and P19 cell lines and significantly increased the susceptibility of testicular cancer to CDDP. Combined with CDDP, MK2206 potentiated CDDP-induced cytotoxicity and apoptosis, with repressed expression of p-Akt and its downstream targets. The subcutaneous xenograft models also showed that a combined CDDP/MK2206 therapy completely suppressed tumor growth without any side effects. CONCLUSION: These results suggested that the concomitant use of MK2206 could enhance the CDDP-induced cytotoxicity and apoptosis in testicular cancer with the suppressed expression of Akt pathway.     

HOXD8 inhibits the proliferation and migration of triple-negative breast cancer cells and induces apoptosis in them through regulation of AKT/mTOR pathway

HOXD8 (Homeobox D8) functions as an apoptotic inducer to suppress tumor progression. However, the role of HOXD8 in triple-negative breast cancer (TNBC) has not been fully understood. Firstly, HOXD8 was found to be reduced in TNBC tissues based on the TCGA samples through Ualcan (http://ualcan.path.uab.edu/analysis.html) prediction. Moreover, data from qRT-PCR and western blot confirmed the lower expression of HOXD8 in the TNBC tissues or cells than that in paracancerous tissues or human mammary epithelial cell line (MCF10A), respectively. Secondly, pcDNA-mediated over-expression of HOXD8 were conducted in TNBC cells, and the gain-of functional assays showed that over-expression of HOXD8 promoted TNBC cell progression with repressed cell apoptosis and induced proliferation, migration and invasion. Moreover, xenografted mouse model was constructed by injection of tumor cell line with stable over-expression of HOXD8 to assess the in vivo tumor growth, and the results revealed that over-expression of HOXD8 inhibited tumor growth. Lastly, our results showed that AKT and mTOR phosphorylation were repressed by HOXD8 over-expression in TNBC cells. In conclusion, HOXD8 functioned as an apoptotic inducer to suppress TNBC cell growth and progression by inhibition of AKT/mTOR pathway.     

Britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by targeting PD-L1 via abrogation of the crosstalk between Myc and HIF-1α in cancer

BackgroundProgrammed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects.PurposeIn this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells.MethodsIn vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model.ResultsBritannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model.ConclusionBritannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.     

Sema4D/Plexin-B1 promotes the progression of osteosarcoma cells by activating Pyk2-PI3K-AKT pathway

Objectives:Osteosarcoma (OS) is one of the two most common malignant bone tumors among children and teens but it is still a rare disorder. Semaphorin 4D (Sema4D) has been reported to play a specific role in human cancers. The aim of this study was to explore the function of Sema4D in the tumorigenesis and development of OS.Methods:10 pairs of OS tissues and paracancerous normal tissues from human OS samples and OS cell lines were used. Western blot assay was performed to detect the protein expression of Sema4D, Plexin-B1, and associated proteins of Pyk2-PI3K/AKT pathway. To explore the effect of Sema4D in the progression of OS, we reduced the expression of Sema4D. The effect of Sema4D knockdown on cell proliferation was explored by CCK-8 assay and clone formation assay. The effect of Sema4D knockdown on cell migration and invasion was assessed by Transwell assay.Results:Sema4D was overexpressed in OS tissues and cell lines. Sema4D knockdown notably suppressed cell proliferation in OS cells. Cell migration and invasion were reduced by Sema4D knockdown. Sema4D/Plexin-B1 facilitated OS, progression by promoting Pyk2-PI3K/AKT pathway.Conclusion:Sema4D/Plexin-B1 promoted the development of OS so Sema4D might be a potential target of treatment for patients with OS.     

Aplysin induces apoptosis in glioma cells through HSP90/AKT pathway

Glioma is one of the most common malignancies in the world. However, an effective regiment is lacking. Increasing evidence indicated that PI3K/AKT signaling is critical for the survival of glioma. In this study, we aimed to study the effect of aplysin on the survival and proliferation of GL26 glioma cells and the involved mechanisms. The data showed that aplysin suppressed the viability of glioma cells in both dose- and time-dependent manners. It also induced G₀/G₁ arrest and apoptosis in glioma cells. Western blot assays revealed that aplysin treatment changed p-AKT expression by impairing the formation of Heat shock protein 90/AKT complex. Aplysin significantly increased the survival time of mice-bearing glioma and reduced the weights of the established gliomas. Collectively, aplysin can inhibit the proliferation of GL26 glioma cells and induce apoptosis in vitro, perhaps through suppressing PI3K/AKT pathway. It can also inhibit glioma growth in vivo and prolong the survival of mice. Thus, aplysin may be a novel therapeutic drug for glioma.     

Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway

BackgroundTriple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia.PurposeThe present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis.Study DesignIn vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasisMethodsCell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters.ResultsLuteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region.ConclusionOur findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.     

Inhibition of the AKT pathway in cholangiocarcinoma by MK2206 reduces cellular viability via induction of apoptosis

Introduction

Cholangiocarcinoma (CCA) is an aggressive disease with limited effective treatment options. The PI3K/Akt/mTOR pathway represents an attractive therapeutic target due to its frequent dysregulation in CCA. MK2206, an allosteric Akt inhibitor, has been shown to reduce cellular proliferation in other cancers. We hypothesized that MK2206 mediated inhibition of Akt would impact CCA cellular viability.

Study methods

Post treatment with MK2206 (0-2 μM), cellular viability was assessed in two human CCA cell lines—CCLP-1 and SG231—using an MTT assay. Lysates from the MK2206 treated CCA cells were then examined for apoptotic marker expression levels using Western blot analysis. Additionally, the effect on cellular proliferation of MK2206 treatment on survivin depleted cells was determined.

Results

CCLP-1 and SG231 viability was significantly reduced at MK2206 concentrations of 0.5, 1, and 2 μM by approximately 44%, 53%, and 64% (CCLP-1; p = 0.01) and 32%, 32%, and 42% (SG231; p < 0.00005) respectively. Western analysis revealed a decrease in AKT^Ser473, while AKT^Thr308 expression was unchanged. In addition, cleaved PARP as well as survivin expression increased while pro-caspase 3 and 9 levels decreased with treatment. Depletion of survivin in CCLP-1 cells resulted in apoptosis as evidenced by increased cleaved PARP. In addition, survivin siRNA further enhanced the antitumor activity of MK2206.

Conclusions

This study demonstrates that by blocking phosphorylation of Akt at serine473, CCA cellular growth is reduced. The growth suppression appears to be mediated via apoptosis. Importantly, combination of survivin siRNA transfection and MK2206 treatment significantly decreased cell viability.     

Dulcitol suppresses proliferation and migration of hepatocellular carcinoma via regulating SIRT1/p53 pathway

BackgroundHepatocellular carcinoma (HCC) spreads further with continuance and increasing incidence due to its high-grade malignancy and metastasis. More effectual strategies on blocking proliferation and metastasis of cancer cells should be studied in HCC. Dulcitol, a natural product extracted from euonymus alatus, was reported that it could induce apoptosis of C6 glioma cells. However, the underlying mechanism of Dulcitol on HCC remains unclea.PurposeIn this study, we aimed to reveal the effect and potential mechanisms of Dulcitol on hepatocellular carcinoma in vitro and in vivo.Study design and methods The cell proliferation and apoptosis were evaluated by MTT, Ki-67 and Hoechst 33258/PI double staining. The migratory and invasive abilities of HepG2 cells were measured by wound-healing and transwell assays. Pathological changes of tumor tissue were observed by HE staining and IHC methods. The expression levels of protein were detected using Western Blot analysis.ResultsThe results showed that Dulcitol inhibited HepG2 cells proliferation by down-regulating the protein expression of SIRT1, Bcl-2, along with up-regulating p53, acetylated-p53 (K382), cleaved-caspase9, cleaved-caspase3, Bax, and cytochrome c in a dose-dependent manner. Furthermore, Dulcitol surpressed the migration and invasion of HepG2 cells through decreasing the levels of MMP-2, uPA and MMP-9 and increasing E-cadherin associated with tumor invasion. In vivo, Dulcitol distinctly inhibited the growth of HepG2 cancer xenograft tumors via inhibiting SIRT1/p53 pathway.ConclusionsOur findings suggested that Dulcitol acted as a SIRT1 inhibitor, inducing apoptosis and inhibiting proliferation, migration and invasion of HepG2 cells and its modulatory mechanism seemed to be associated with regulation of MMPs, SIRT1/p53 pathways.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.