Propofol Ameliorates ox-LDL-Induced Endothelial Damage Through Enhancing Autophagy via PI3K/Akt/m-TOR Pathway: A Novel Therapeutic Strategy in Atherosclerosis

Objective: Atherosclerosis (AS) represents a common age-associated disease, which may be accelerated by oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell injury. This study aimed to investigate the effects of Propofol on ox-LDL-induced endothelial damage and the underlying molecular mechanisms.Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to ox-LDL to induce endothelial damage. HUVECs were pretreated with 0, 5, 25 and 100°μM Propofol, followed by exposure to 100°μg/ml ox-LDL for 24°h. Cell viability was assessed by cell counting kit-8 (CCK-8) assay. The expression of autophagy- and apoptosis-related proteins was detected via western blot. Autophagosome was investigated under a transmission electron microscope. After co-treatment with autophagy inhibitor Bafilomycin A1 or si-Beclin-1, cell apoptosis was detected by flow cytometry. Furthermore, under cotreatment with PI3K activator 740Y-P, PI3K/Akt/m-TOR pathway- and autophagy-related proteins were examined by western blot.Results: With a concentration-dependent manner, Propofol promoted the viability of HUVECs exposed to ox-LDL, and increased LC3-II/I ratio and Beclin-1 expression, and decreased P62 expression. The formation of autophagosome was enhanced by Propofol. Furthermore, Propofol treatment elevated Bcl-2/Bax ratio and lowered Caspase-3 expression. Bafilomycin A1 or si-Beclin-1 distinctly ameliorated the inhibitory effects of Propofol on apoptosis in ox-LDL-exposed HUVECs. Moreover, Propofol lowered the activation of PI3K/Akt/m-TOR pathway in HUVECs under exposure to ox-LDL. However, its inhibitory effects were weakened by 740Y-P.Conclusion: Collectively, this study revealed that Propofol could ameliorate ox-LDL-induced endothelial damage through enhancing autophagy via PI3K/Akt/m-TOR pathway, which might offer a novel therapeutic strategy in AS.

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Knockdown of BAG3 synergizes with olaparib to kill ovarian cancer cells via repressing autophagy

Journal of Investigative Medicine ◽

10.1136/jim-2020-001602 ◽

2020 ◽

pp. jim-2020-001602

Author(s):

Kexin Wang ◽

Jianhua Zheng

Keyword(s):

Ovarian Cancer ◽

Western Blot ◽

Transfection Efficiency ◽

Parp Inhibitor ◽

Beclin 1 ◽

Cell Counting ◽

Ovarian Cancer Cells ◽

Western Blot Assay ◽

Related Proteins

This study aimed at expounding the synergistic effect of Bcl-2-associated athanogene 3 (BAG3) knockdown and poly ADP-ribose polymerase (PARP) inhibitor on ovarian cancer (OC) cells and the potential mechanism. Short hairpin RNA (shRNA) targeting BAG3 (sh-BAG3) was transfected into SK-OV-3 (SKOV-3 ;SKOV3) and A2780 cells, and western blot assay was used to detect transfection efficiency. Cell proliferation and apoptosis were detected by the cell counting kit-8 method, 5-Bromodeoxyuridine (BrdU) experiment and flow cytometry analysis, respectively. The expressions of apoptosis-related proteins Bax and Bcl-2, as well as the expressions of autophagy-related proteins LC3-I, LC3-II and Beclin-1, were examined by western blot assay. Additionally, the cells were treated with autophagy activator rapamycin to investigate whether the tumor-suppressive function of BAG3 knockdown+PARP inhibitor was dependent on autophagy. In this work, we demonstrated that BAG3 knockdown further sensitized OC cells to olaparib treatment, reducing cellular viability and promoting apoptosis. Both sh-BAG3 and olaparib decreased the expression of Beclin-1 and the LC3-Ⅱ:LC3-I ratio, and their synergism further inhibited the process of autophagy. However, the aforementionede effects were reversed after the cells were treated with rapamycin. Based on these results, we concluded that BAG3 knockdown synergizes with olaparib to kill OC cells in vitro by repressing autophagy.

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Novel Therapeutic Strategy Targeting NHE1 and Its Upstream Activators in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v124.21.913.913 ◽

2014 ◽

Vol 124 (21) ◽

pp. 913-913

Author(s):

Cheuk-Him Man ◽

Chae-Yin Cher ◽

Stephen S.Y. Lam ◽

Eric S.K. Ho ◽

Nelson K.L. Ng ◽

...

Keyword(s):

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Therapeutic Strategy ◽

Inhibitory Effects ◽

Growth Inhibitory ◽

Specific Inhibitors ◽

Acute Myeloid ◽

Novel Therapeutic

Abstract Increase in Tescalcin (TESC) gene expression and intracellular pH (pHi) have been associated with drug resistance in acute myeloid leukemia (AML). Tescalcin was shown to stabilize the membrane sodium/hydrogen exchanger (NHE1) that maintains a high pHi by H+ efflux in exchange for Na+. NHE1 has also been shown to be activated by PDGFR, PKC, calmodulin, p90-RSK and ROCK-RhoA, but their relevance to leukemogenesis and drug resistance in AML was unknown. We hypothesized that targeting NHE1 and its upstream activators might offer a novel and effective therapeutic strategy in AML. AML cell lines and mononuclear cell fraction from peripheral blood (PB) or bone marrow (BM) of AML patients (comprising primarily myeloblasts as shown by microscopic review of cytospin preparations) were treated with inhibitors for 3 days (concentrations: 0.1nM to 10mM) that target potential activators of NHE1. The anti-leukemia effects of these inhibitors were evaluated by PrestoBlue® Cell Viability Reagent as a measure of viable cell number. Their effects on pHi and apoptosis were evaluated by SNARF-1 and Annexin V/7-AAD staining respectively by flow cytometry. AML cell lines ML2, Kasumi-1, MOLM-13 and MV4-11 (IC50 in mM: 12.2, 13.1, 11.6 and 9.2 respectively) were more sensitive than KG1, NB4, THP-1 and OCI-AML3 (IC50 in mM: 30.7, 24.8, 119.2 and 49.4 respectively) to the growth inhibitory effects of NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA), accompanied with a larger extent of cellular acidification and apoptosis induction in those 4 HMA-sensitive lines. To look for the upstream activators of NHE1 relevant to AML, the cell lines were treated with specific inhibitors targeting potential NHE1 activators. Both HMA-sensitive and insensitive cell lines were susceptible to the intracellular acidification and growth inhibition by PDGFR and p90-RSK inhibitors. Furthermore, FLT3 inhibitors, sorafenib and quizartinib, also reduced pHi of FLT3-ITD+ (Fms-Like Tyrosine Kinase 3 - Internal Tandem Duplication) AML cell lines, MOLM-13 and MV4-11, suggesting that FLT3-ITD might also activate NHE1, resulting in high pHi of FLT3-ITD+ AML. Different primary AML samples were treated with inhibitors to NHE1 (n=50), PDGFR (n=50) and p90-RSK (n=36) (Concentration: 100nM to 10mM) in vitro. Their response to the growth inhibitory effect of HMA, accompanied by effective pHi reduction (n=10), correlated with that of PDGFR and p90-RSK inhibitors (Pearson r=0.74, p<0.001 and r=0.73, p<0.001 respectively), supporting the proposition that these signaling pathways might be the critical and common activators of NHE1. Synergism of anti-leukemia effects could also be demonstrated between HMA and PDGFR inhibitors, calculated by Excess over Bliss Additivism (EOBA). To evaluate the clinical relevance of the study, serum was obtained from medical patients treated with high dose amiloride (20 mg daily), an NHE1 inhibitor, for underlying congestive heart failure. Compared with the serum of healthy volunteers, the amiloride-containing serum significantly reduced the pHi (n=10, p=0.001), induced apoptosis (n=4, p=0.04) and potentiated the inhibitory effects of PDGFR inhibitors (n=4, p=0.04) in primary AML samples. NHE1 might be a potential target in drug-resistant AML and activated by PDGFR, PKC, p90-RSK or both in a patient-specific fashion. Therefore, employing specific inhibitors to target NHE1 and its upstream activators should be explored as novel therapeutic strategy in this group of patients. Disclosures No relevant conflicts of interest to declare.

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Expression of Autophagy-Related Proteins in Hürthle Cell Neoplasm Is Different from That in Follicular Neoplasm

Disease Markers ◽

10.1155/2017/1372387 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yoon Jin Cha ◽

Hye Min Kim ◽

Ja Seung Koo

Keyword(s):

Western Blot ◽

Immunohistochemical Staining ◽

Beclin 1 ◽

Follicular Neoplasm ◽

Clinical Implications ◽

Positive Staining ◽

Hürthle Cell ◽

Cell Neoplasm ◽

Expression Rate ◽

Related Proteins

Purpose. We aimed to evaluate expression of autophagy-related proteins in Hürthle cell neoplasm (HCN) and follicular neoplasm (FN) and assess the clinical implications. Methods. 265 FNs (112 follicular carcinomas and 153 follicular adenomas) and 108 HCNs (27 Hürthle cell carcinomas and 81 Hürthle cell adenomas) were made into a tissue microarray. Immunohistochemical staining and Western blot for autophagy-related proteins (beclin-1, light chain (LC) 3A, LC3B, p62, and BNIP3) were performed, and the results were statistically analyzed. Results. A higher expression rate of beclin-1, LC3B, p62, and BNIP3 was found in HCN than in FN (P<0.001). The expression rate of beclin-1, LC3B, p62, and BNIP3 was the highest in HCCs followed by HCAs, FCs, and FAs in that order (P<0.001). HCCs were positive for the largest number of autophagy-related proteins followed by HCAs, FCs, and FAs (P<0.001), and most of the positive markers identified in HCCs were the high autophagy type (P<0.001), defined by positive staining for three or more of the five autophagy-related proteins. Conclusion. The autophagy-related proteins, beclin-1, LC3A, LC3B, p62, and BNIP3, were more frequently expressed in HCNs than in FNs, and HCCs showed the highest expression rate.

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The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-020-02056-z ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Jin Tao ◽

Hui Chen ◽

Xiaolei Li ◽

Jingcheng Wang

Keyword(s):

Transcription Factor ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cell Counting ◽

Immunofluorescent Staining ◽

Fibroblast Proliferation ◽

Activating Transcription Factor ◽

Related Proteins

Abstract Background The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. Methods The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. Results It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. Conclusion Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.

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Cancer Associated Fibroblast-Derived IL-6 Determines Unfavorable Prognosis in Cholangiocarcinoma By Affecting Autophagy-Associated Chemoresponse

10.21203/rs.3.rs-117710/v1 ◽

2020 ◽

Author(s):

Suyanee Thongchot ◽

Chiara Vidoni ◽

Alessandra Ferraresi ◽

Watcharin Loilome Loilome ◽

Narong Khuntikeo ◽

...

Keyword(s):

Cancer Cells ◽

Therapeutic Strategy ◽

Molecular Data ◽

Cell Counting ◽

Effective Response ◽

Mrna Expression Data ◽

Cancer Associated Fibroblast ◽

Related Proteins ◽

Invasiveness Potential

Abstract Background: Interleukin-6 (IL-6) massively released by cancer-associated fibroblast (CAFs) has been shown to associate with the malignant behavior of cholangiocarcinoma (CCA). In vitro studies demonstrated the ability of CAFs-derived IL-6 to inhibit autophagy in CCA cells thus promoting their proliferation and invasiveness potential. Here, we aimed to validate with clinical and molecular data the hypothesis that CAFs infiltration and release of IL-6 predict poor prognosis in CCA patients following dysregulation of autophagy in cancer cells.Methods: Stromal IL-6 and cancer cell-associated autophagy proteins LC3 and p62 were assayed by Tissue MicroArray immunohistochemistry and their expression correlated with overall survival (OS) in a cohort of 70 CCA patients. Additionally, copy number and mRNA expression data of BECN1, MAP1-LC3B, p62/SQSTM1 and IL6 were extracted from a CCA database in TCGA and correlated with OS. 5-FU Cytotoxicity in CCA cells was assessed by cell counting, clonogenic assay, cytofluorometry and western blotting and immunofluorescence of apoptotic-related proteins. Results: We show that patients bearing a CCA with low production of stromal IL-6 and active autophagy flux in the cancer cells have the best prognosis and this correlates with a more effective response to post-operative chemotherapy. Similar trend was observed in CCA patients from TCGA database. In vitro experiments with primary CAFs isolated from human CCA and epigenetic manipulations showed that IL-6 plays a pivotal role in determining the autophagy-associated apoptotic response to chemotherapeutic drug in cultured human CCA cells. Conclusions: Our data support a therapeutic strategy that includes autophagy-enhancing drugs along with adjuvants limiting the stromal inflammation (i.e., the secretion of IL-6) to improve the survival of CCA patients.

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Interplay Between Autophagy and Apoptosis in Lycorine Hydrochloride-Induced Cytotoxicity of HCT116 Cells

Natural Product Communications ◽

10.1177/1934578x19862100 ◽

2019 ◽

Vol 14 (7) ◽

pp. 1934578X1986210

Author(s):

Ganting Zhao ◽

Yanjing Wang ◽

Changqing Yang ◽

Li Zhao ◽

Lili Guo ◽

...

Keyword(s):

Western Blot ◽

Colony Formation ◽

Quantitative Polymerase Chain Reaction ◽

Adenosine Monophosphate ◽

Beclin 1 ◽

Cell Counting ◽

Colorectal Cancer Cell Line ◽

Rna Transfection ◽

Induced Apoptosis ◽

Hct116 Cells

The aim of this study was to investigate the antitumor effect of lycorine hydrochloride (LH) and discuss the correlation between LH-induced apoptosis and autophagy in the human colorectal cancer cell line HCT116. Here the results by the Cell Counting Kit-8 and colony formation assays showed that LH concentration-dependently decreased cell viability and colony formation in HCT116 cells, suggesting inhibition of cell proliferation by LH. By flow cytometry, LH was found to increase apoptotic rate in HCT116 cells. Mechanistically, Western blot results revealed that LH increased the expression of the protein of Bax and Caspase-3, and decreased Bcl-2 proteins expression. Moreover, the reverse transcriptase quantitative polymerase chain reaction and Western blot analysis also showed that LH increased the expression of Beclin-1 and LC3B-II/LC3B-I ratio, indicating that autophagy was induced by LH. LH induced autophagy via downregulating phospho-mammalian target of rapamycin and upregulating phospho-AMPK (5′ adenosine monophosphate-activated protein kinase). Furthermore, to understand the role of LH-induced autophagy and its association with apoptosis, cells were analyzed after Beclin-1 small interfering RNA transfection. The results indicated that the proapoptotic ability of LH was increased by inhibition of autophagy. In conclusion, the present investigation suggested that LH induced apoptosis and autophagy in HCT116 cells via the mitochondrial and AMPK/mTOR pathways. The suppression of autophagy promoted LH-induced apoptosis by modulating Beclin-1 and Bcl-2.

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Long Non-coding RNA EBLN3P Regulates UHMK1 Expression by Sponging miR-323a-3p and Promotes Colorectal Cancer Progression

Frontiers in Medicine ◽

10.3389/fmed.2021.651600 ◽

2021 ◽

Vol 8 ◽

Author(s):

Xiang-hao Xu ◽

Wen Song ◽

Jun-hua Li ◽

Ze-qi Huang ◽

Ya-fang Liu ◽

...

Keyword(s):

Cancer Progression ◽

Cell Counting ◽

Inhibitory Effects ◽

Poor Clinical Outcome ◽

Non Coding Rna ◽

Proliferation And Metastasis ◽

Polymerase Chain ◽

Colorectal Cancer Progression ◽

Related Proteins ◽

Long Non Coding Rna

Background: Growing studies have demonstrated that long non-coding RNA (lncRNA) can act as crucial roles during the progression of various tumors, including colorectal carcinoma (CRC). We aimed to determine lncRNA endogenous bornavirus-like nucleoprotein (EBLN3P) expression in CRC and examine its influence on tumor behaviors of CRC cells.Materials and Methods: Quantitative real-time polymerase chain reaction was used to determine the expression levels of EBLN3P and miR-323a-3p in CRC tissues and cell lines. Cell viability, migration, invasion, and apoptosis were assessed by Cell Counting Kit 8, colony formation, Transwell assay, wound healing assays, and flow cytometry. Bioinformatics and dual-luciferase assays were used to investigate the interaction between EBLN3P and miR-323a-3p, as well as between miR-323a-3p and U2AF homology motif kinase 1 (UHMK1). Western blot was applied for detecting the expressions of the related proteins.Results: EBLN3P was highly expressed in CRC, and its high expression was distinctly associated with increased tumor size, histology/differentiation and advanced TNM stage, and poor clinical outcome of CRC patients. EBLN3P silencing significantly inhibited the proliferation and metastasis and induced the apoptosis of CRC cells. Mechanistically, overexpression of EBLN3P exhibited tumorigenic effects through downregulating the inhibitory effects of miR-323a-3p on UHMK1 expression. The correlation analysis confirmed the positive or negative association among EBLN3P, miR-323a-3p, and UHMK1.Conclusion: EBLN3P promoted the development of CRC via targeting miR-323a-3p/UHMK1, which provided a new idea for treating CRC.

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Anti-OX40ligand treatment as a novel therapeutic strategy in a murine model of colitis

Gastroenterology ◽

10.1016/s0016-5085(01)83410-7 ◽

2001 ◽

Vol 120 (5) ◽

pp. A685-A685

Author(s):

B SINGH ◽

V MALMSTROM ◽

F POWRIE

Keyword(s):

Murine Model ◽

Therapeutic Strategy ◽

Novel Therapeutic

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Anticancer Immunotoxins, Challenges, and Approaches

Current Pharmaceutical Design ◽

10.2174/1381612826666201006155346 ◽

2020 ◽

Vol 26 ◽

Author(s):

Maryam Dashtiahangar ◽

Leila Rahbarnia ◽

Safar Farajnia ◽

Arash Salmaninejad ◽

Arezoo Gowhari Shabgah ◽

...

Keyword(s):

Therapeutic Strategy ◽

Antibody Fragment ◽

Clinical Use ◽

Multiple Cancer ◽

Main Obstacle ◽

Cancer Types ◽

Recombinant Immunotoxins ◽

Cancerous Cells ◽

High Immunogenicity ◽

Novel Therapeutic

: The development of recombinant immunotoxins (RITs) as a novel therapeutic strategy has made a revolution in the treatment of cancer. RITs are resulting from the fusion of antibodies to toxin proteins for targeting and eliminating cancerous cells by inhibiting protein synthesis. Despite indisputable outcomes of RITs regarding inhibiting multiple cancer types, high immunogenicity has been known as the main obstacle in the clinical use of RITs. Various strategies have been proposed to overcome these limitations, including immunosuppressive therapy, humanization of the antibody fragment moiety, generation of immunotoxins originated from endogenous human cytotoxic enzymes, and modification of the toxin moiety to escape the immune system. This paper devoted to reviewing recent advances in the design of immunotoxins with lower immunogenicity.

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