scholarly journals Circumventing AKT-Associated Radioresistance in Oral Cancer by Novel Nanoparticle-Encapsulated Capivasertib

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 533 ◽  
Author(s):  
Liwei Lang ◽  
Tiffany Lam ◽  
Alex Chen ◽  
Caleb Jensen ◽  
Leslie Duncan ◽  
...  
Keyword(s):  
3D Culture ◽  
Small Animal ◽  
Tumor Model ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Therapeutic Effects ◽  
Akt Inhibitor ◽  
Orthotopic Mouse Model ◽  
Improvement In Survival

Background: Development of radioresistance in oral squamous cell carcinoma (OSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in recent decades. Effective strategies to overcome radioresistance are necessary to improve the therapeutic outcomes of radiotherapy in OSCC patients. Methods: Cells and xenograft tumors were irradiated using the Small Animal Radiation Research Platform. AKT inhibitor capivasertib (AZD5363) was encapsulated into cathepsin B-responsible nanoparticles (NPs) for tumor-specific delivery. Cell viability was measured by alamarBlue, cell growth was determined by colony formation and 3D culture, and apoptosis was assessed by flow cytometry with the staining of Fluorescein isothiocyanate (FITC) Annexin V and PI. An orthotopic tongue tumor model was used to evaluate the in vivo therapeutic effects. The molecular changes induced by the treatments were assessed by Western blotting and immunohistochemistry. Results: We show that upregulation of AKT signaling is the critical mechanism for radioresistance in OSCC cells, and AKT inactivation by a selective and potent AKT inhibitor capivasertib results in radiosensitivity. Moreover, relative to irradiation (IR) alone, IR combined with the delivery of capivasertib in association with tumor-seeking NPs greatly enhanced tumor cell repression in 3D cell cultures and OSCC tumor shrinkage in an orthotopic mouse model. Conclusions: These data indicate that capivasertib is a potent agent that sensitizes radioresistant OSCC cells to IR and is a promising strategy to overcome failure of radiotherapy in OSCC patients.

scholarly journals TRIM32 Promotes the Growth of Gastric Cancer Cells through Enhancing AKT Activity and Glucose Transportation

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Jianjun Wang ◽  
Yuejun Fang ◽  
Tao Liu
Keyword(s):  
Gastric Cancer ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Cell Counting ◽  
Akt Inhibitor ◽  
Gastric Cancer Cells ◽  
Poor Overall Survival ◽  
Reverse Transcription Pcr ◽  
Apoptosis Detection

Tripartite motif protein 32 (TRIM32), an E3 ubiquitin ligase, is a member of the TRIM protein family. However, the underlying function of TRIM32 in gastric cancer (GC) remains unclear. Here, we aimed to explore the function of TRIM32 in GC cells. TRIM32 was induced silencing and overexpression using RNA interference (RNAi) and lentiviral-mediate vector in GC cells, respectively. Moreover, the PI3K/AKT inhibitor LY294002 was used to examine the relationship between TRIM32 and AKT. Quantitative reverse-transcription PCR (qRT-PCR) and western blot were used to determine the mRNA and protein contents. The glucose analog 2-NBDG was used as a fluorescent probe for determining the activity of glucose transport. An annexin V-fluorescein isothiocyanate apoptosis detection kit was used to stain NCI-N87, MKN74, and MKN45 cells. Cell counting kit-8 (CCK-8) assay was used to examine cell proliferation. Our results indicated that TRIM32 was associated with poor overall survival of patients with GC. Moreover, TRIM32 was a proproliferation and antiapoptosis factor and involved in the AKT pathway in GC cells. Furthermore, TRIM32 possibly mediated the metabolism of glycolysis through targeting GLUT1 and HKII in GC cells. Importantly, TRIM32 silencing deeply suppressed the tumorigenicity of GC cells in vivo. Our findings not only enhanced the understanding of the function of TRIM32 but also indicated its potential value as a target in GC treatment.

scholarly journals Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Xuejie Jiang ◽  
Ling Jiang ◽  
Jiaying Cheng ◽  
Fang Chen ◽  
Jinle Ni ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Histone Deacetylases ◽  
Myeloid Leukemia ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Gli 1 ◽  
Acute Myeloid

Abstract Background Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. Methods Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. Results In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. Conclusions Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
S G Schwab ◽  
K Sarnow ◽  
E Alme ◽  
R Goldbrunner ◽  
H Bjørsvik ◽  
...  
Keyword(s):  
Imaging System ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Therapeutic Effects ◽  
Cell Viability Assay ◽  
Selective Cytotoxicity ◽  
Viability Assay

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

Panax notoginseng Inhibits Tumor Growth through Activating Macrophage to M1 Polarization

2018 ◽  
Vol 46 (06) ◽  
pp. 1369-1385 ◽  
Author(s):  
Bosung Kim ◽  
Eun-Yeong Kim ◽  
Eun-Ji Lee ◽  
Jung Ho Han ◽  
Chung-Hwan Kwak ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Culture Media ◽  
Tumor Model ◽  
Annexin V ◽  
Panax Notoginseng ◽  
Raw264.7 Cells ◽  
M1 Polarization ◽  
Chemotactic Protein ◽  
M1 Phenotype

Among the herbal ingredients of HangAmDan-B, a medicinal formula that redirects macrophages to become tumoricidal effectors, we found that Panax notoginseng (Burk.) F. H. Chen is the active component responsible for its macrophage-mediated antitumor activity. The water extracted roots of P. notoginseng (PN) did not affect the viability of RAW264.7 murine macrophage-like cells and murine Lewis lung carcinoma (LLC) cells up to a concentration of 100[Formula: see text][Formula: see text]g/mL. However, the transfer of culture media from PN-treated RAW264.7 cells suppressed the growth of LLC cells. The expression of classically activated (M1) markers, such as interleukin (IL)-1[Formula: see text], monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-[Formula: see text], and inducible nitric oxide synthase (iNOS), was increased by PN treatment. The expression of alternatively activated (M2) markers including CD206, IL-10, and [Formula: see text]-[Formula: see text]-acetylhexosaminidases (YM-1) was reduced by PN treatment in the presence of IL-4. Flow cytometry also revealed that PN drives M1 activation of RAW264.7 cells. The transfer of culture media from PN-treated RAW264.7 cells induced the apoptosis of LLC cells as measured by flow cytometry using Annexin-V staining and western blot analysis for caspase cascade-related proteins. In addition, the results from in vivo tumor allograft model demonstrated that PN reduced both tumor volume and weight. The activation of macrophages toward an M1 phenotype was confirmed in the tumor allograft tumor model. These results collectively show that PN can serve as a potent anticancer agent through reeducation of macrophages toward an M1 phenotype.

Phenotypic and Functional Effects of Perifosine on Dendritic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4803-4803
Author(s):  
Weihua Song ◽  
Teru Hideshima ◽  
Yu-Tzu Tai ◽  
Kenneth C. Anderson ◽  
Nikhil C. Munshi
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Annexin V ◽  
Antitumor Agent ◽  
Dependent Manner ◽  
Immune Functions ◽  
Akt Inhibitor ◽  
Antitumor Effects

Abstract Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo mouse model. Currently perifosine is under the evaluation of phase II clinical trail in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. The objective of this study is to evaluate the effects of perifosine on the activity of antigen presenting cells (APCs). Monocyte-derived dendritic cells (DCs) from normal human donors were used as the APCs, and mature DCs were obtained by the treatment of TNF-α and IL-1β. Perifosine was used at the concentrations of 2.5 uM, 5 uM and 10 uM for the treatment with DCs. We first evaluated the effect of perifosine on the survival of DCs. We observed that the perifosine treatment up to 48 hours had no effect on viability (>90%) of DCs, assessed by annexin V and PI staining. Alteration of phenotype by perifosine on DCs was further examined by flow cytometry. Our results demonstrated that with dose-dependent manner, perifosine led to a significant down-regulation of surface antigens on immature DCs at 24 and 48 hours, which associated to costimulation (CD40, CD80 and CD86), antigen presentation (HLA-ABC, HLA-DPQR) and maturation (CD83). However, we did not observed significant effect of perifosine on above surface markers on mature DCs. Since DCs play a crucial role on the regulation of Th1/Th2 immune responses by the production of IL-12, we next evaluated IL-12 secretion by DCs with and without perifosine treatment. Importantly, treatment with perifosine significantly decreased LPS-induced-IL-12 production, compared to untreated DCs (untrt vs. trt = 192.29 vs. 166.23 pg/ml (2.5uM), 111.19 pg/ml (5uM) and 44.886 pg/ml (10uM)) at 24 hours. To assess the effect of perifosine on DCs function on the regulation of T cell responses, we stimulated allogenic T cells with mature DCs with or without the pre-treatment of perifosine. The proliferation assay by 3H-TdR incorporation and IFN-γ production by ELISA indicated perifosine-treated DCs had no significant effect on the regulation of T cells function. Taken together, these results showed that DCs function are influenced by the treatment of perifosine. Our pre-clinical data therefore indicates the need to monitor immune functions in patients under the Akt inhibitor treatment.

scholarly journals Sox9-expressing cells promote regeneration after radiation-induced lung injury via PI3K/AKT pathway

2021 ◽  
Author(s):  
Shuang Chen ◽  
Kang Li ◽  
Xinqi Zhong ◽  
Ganping Wang ◽  
Xiaocheng Wang ◽  
...  
Keyword(s):  
Lung Injury ◽  
Background Radiation ◽  
Lineage Tracing ◽  
Akt Pathway ◽  
Therapeutic Effects ◽  
Akt Inhibitor ◽  
Thoracic Radiation ◽  
Radiation Induced ◽  
Epithelium Regeneration

Abstract Background Radiation induced lung injury (RILI) is considered as one of the most common complications of thoracic radiation. Recent studies have focused on stem cells properties to obtain ideal therapeutic effects and Sox9 has been reported to be involved in stem cell induction and differentiation. However, whether Sox9-expressing cells play a role in radiation repair and regeneration remain unknown. Methods We successfully obtain SOX9CreER, RosatdTomato and RosaDTA mice and identify Sox9-expressing cells through lineage tracing assay. Then we evaluated the effects of the ablation of Sox9-expressing cells in vivo. Furthermore, we investigated the underlying mechanism of Sox9 expressing cells during lung regeneration via an online single cell RNA-seq dataset. Results In our study, we demonstrated Sox9-expressing cells promote regenerative of lung tissues and ablation of Sox9-expressing cells leads to severe phenotypes after radiation damage. In addition, analysis of online scRNA-seq dataset revealed an enrichment of PI3K/AKT pathway in Sox9-expressing cells during lung epithelium regeneration. Finally, AKT inhibitor Perifosine could suppress the regenerative effects of Sox9-expressing cells. Conclusions Taken together, our study suggests that Sox9-expressing cells may serve as a therapeutic target in the setting of lung tissue after RILI.

scholarly journals Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties

2022 ◽  
Vol 23 (2) ◽  
pp. 863
Author(s):  
Alessia Gallo ◽  
Nicola Cuscino ◽  
Flavia Contino ◽  
Matteo Bulati ◽  
Mariangela Pampalone ◽  
...  
Keyword(s):  
Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Therapeutic Effects ◽  
Rna Seq ◽  
Human Amnion ◽  
Cell Based Therapy ◽  
Therapeutic Properties ◽  
Stromal Stem Cells

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

Targeted Inactivation of Murine Band 3 (AE1) Gene Produces a Hypercoagulable State Causing Widespread Thrombosis In Vivo

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1785-1792 ◽  
Author(s):  
Hani Hassoun ◽  
Ying Wang ◽  
John Vassiliadis ◽  
Mohini Lutchman ◽  
Jiri Palek ◽  
...  
Keyword(s):  
Specific Binding ◽  
Phospholipid Composition ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Band 3 ◽  
Hypercoagulable State ◽  
Outer Leaflet ◽  
Large Vein ◽  
American Society

Abstract Only 5% to 10% of band 3 null mice survive the neonatal period. To determine the cause of death, 3 adult and 11 newborn band 3 null mice were submitted for histopathologic examination. All but 1 pup showed evidence of thrombosis including: (1) large thrombotic lesions in the heart, which were partially organized, calcified in some fields, and endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver suggestive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate the etiology of this hypercoagulable state, we have used the Russell’s viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no effect, suggesting that the membrane of band 3 null RBCs provides a suitable surface for activation of the prothrombinase complex. Using flow cytometry, we have examined the phosphatidylserine (PS)-specific binding of fluorescein isothiocyanate (FITC)-annexin V to normal and band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as compared with normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS exposure on the outer membrane leaflet. These findings are further supported by direct fluorescence microscopy of normal and band 3 null RBCs labeled with FITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable state, which appears to originate from changes in the phospholipid composition of the membrane leading to PS exposure on the outer leaflet. © 1998 by The American Society of Hematology.

scholarly journals Real-Time Fluorescence Imaging Using Indocyanine Green to Assess Therapeutic Effects of Near-Infrared Photoimmunotherapy in Tumor Model Mice

2020 ◽  
Vol 19 ◽  
pp. 153601212093496
Author(s):  
Adrian Rosenberg ◽  
Daiki Fujimura ◽  
Ryuhei Okada ◽  
Aki Furusawa ◽  
Fuyuki Inagaki ◽  
...  
Keyword(s):  
Indocyanine Green ◽  
Real Time ◽  
Fluorescence Imaging ◽  
Near Infrared ◽  
Imaging System ◽  
Tumor Model ◽  
Therapeutic Effects ◽  
Tumor Perfusion ◽  
Background Ratio

Background: Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that causes an increase in tumor perfusion, a phenomenon termed the super-enhanced permeability and retention effect. Currently, in vivo treatment efficacy of NIR-PIT is observable days after treatment, but monitoring would be improved by more acute detection of intratumor change. Fluorescence imaging may detect increased tumor perfusion immediately after treatment. Methods: In the first experiment, athymic nude mouse models bearing unilateral subcutaneous flank tumors were treated with either NIR-PIT or laser therapy only. In the second experiment, mice bearing bilateral flank tumors were treated with NIR-PIT only on the left-sided tumor. In both groups, immediately after treatment, indocyanine green was injected at different doses intravenously, and mice were monitored with the Shimadzu LIGHTVISION fluorescence imaging system for 1 hour. Results: Tumor-to-background ratio of fluorescence intensity increased over the 60 minutes of monitoring in treated mice but did not vary significantly in control mice. Tumor-to-background ratio was highest in the 1 mg kg−1 and 0.3 mg kg−1 doses. In mice with bilateral tumors, tumor-to-untreated tumor ratio increased similarly. Conclusions: Acute changes in tumor perfusion after NIR-PIT can be detected by real-time fluorescence imaging.

scholarly journals Disappearance of Vimentin in Sertoli Cells: A Mono(2-ethylhexyl) Phthalate Effect

2007 ◽  
Vol 26 (4) ◽  
pp. 289-296 ◽  
Author(s):  
Tat Wei Tay ◽  
Bibin Bintang Andriana ◽  
Maki Ishii ◽  
Naoki Tsunekawa ◽  
Yoshiakira Kanai ◽  
...  
Keyword(s):  
Sertoli Cell ◽  
Cell Cultures ◽  
Sertoli Cells ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Blue Staining ◽  
Spermatogenic Cells ◽  
Gradual Disappearance ◽  
Ethylhexyl Phthalate

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V–FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V–FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

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