Identification of Fangchinoline as a novel autophagy inhibitor with an adjuvant of chemotherapy against lung cancer.

Autophagy is a fundamental recycling pathway that enhances cellular resilience, promoting survival. However, this survival mechanism can impede anti-cancer treatment strategies designed to induce cell death. In this study, we identified a novel autophagy inhibitor, Fangchinoline (Fan) isolated from the traditional Chinese medicine Stephania tetrandra. We speculated that when Fan blocks autophagy, cancer cells lose substantial self-preservation abilities during treatment. Firstly, we examined in detail the mechanism through which Fan inhibits autophagy. Specifically, Fan induced a significant increase in autophagosomes, as indicated by GFP-LC3 labeling, confirmed by the up-regulation of LC3-II. The autophagy receptor protein p62 was also up-regulated, suggesting a potential inhibition of autophagy flux. We further ruled out the possibility of fusion barriers between lysosomes and autophagosomes, as confirmed by their co-localization in double fluorescence staining. However, the lysosomal acid environment might be compromised, as suggested by the diminished fluorescence of acidity-sensitive dyes in the lysosomes and the corresponding decrease in mature forms of lysosomal cathepsin. To test the anti-cancer potential of Fan, we combined it with Cisplatin (Cis) or Paclitaxel (PTX) for lung cancer cell treatment. This combined treatment demonstrated a synergistically enhanced killing effect. These promising anti-tumor results were also replicated in a xenografted tumor model. The significance of this research lies in the identification of Fan as a potent autophagy inhibitor and its potential to enhance the efficacy of existing anti-cancer drugs. By unraveling the mechanisms of Fan's action on autophagy and demonstrating its synergistic effect in combination therapies, our study provides valuable insights for developing novel strategies to overcome autophagy-mediated resistance in cancer treatment.

Identification of Fangjihuangqi Decoction as a late-stage autophagy inhibitor with an adjuvant anti-tumor effect against non-small cell lung cancer.

BACKGROUND: Clinically, although chemotherapy is one of the most commonly used methods of treating tumors, chemotherapeutic drugs can induce autophagic flux and increase tumor cell resistance, leading to drug tolerance. Therefore, theoretically, inhibiting autophagy may improve the efficacy of chemotherapy. The discovery of autophagy regulators and their potential application as adjuvant anti-cancer drugs is of substantial importance. In this study, we clarified that Fangjihuangqi Decoction (FJHQ, traditional Chinese medicine) is an autophagy inhibitor, which can synergistically enhance the effect of cisplatin and paclitaxel on non-small cell lung cancer (NSCLC) cells. METHODS: We observed the changes of autophagy level in NSCLC cells under the effect of FJHQ, and verified the level of the autophagy marker protein and cathepsin. Apoptosis was detected after the combination of FJHQ with cisplatin or paclitaxel, and NAC (ROS scavenger) was further used to verify the activation of ROS-MAPK pathway by FJHQ. RESULTS: We observed that FJHQ induced autophagosomes in NSCLC cells and increased the levels of P62 and LC3-II protein expression in a concentration- and time-gradient-dependent manner, indicating that autophagic flux was inhibited. Co-localization experiments further showed that while FJHQ did not inhibit autophagosome and lysosome fusion, it affected the maturation of cathepsin and thus inhibited the autophagic pathway. Finally, we found that the combination of FJHQ with cisplatin or paclitaxel increased the apoptosis rate of NSCLC cells, due to increased ROS accumulation and further activation of the ROS-MAPK pathway. This synergistic effect could be reversed by NAC. CONCLUSION: Collectively, these results demonstrate that FJHQ is a novel late-stage autophagy inhibitor that can amplify the anti-tumor effect of cisplatin and paclitaxel against NSCLC cells.

A five-parameter score for predicting saphenous vein graft degenerative and/or occlusive disease in recurring ischemic symptoms after one year post coronary artery bypass grafting.

BACKGROUND: The recurrence rate of ischemic symptoms after coronary artery bypass grafting (CABG) is increasing in recent years. How to prevent and treat saphenous vein graft disease (SVGD [symptomatic ⩾50% stenosis in at least one Saphenous vein graft]) has been a clinical challenge to date. Different pathogenesis may exist in SVGD of different periods. There are currently few available scores for estimating the risk of SVGD after one year post CABG. OBJECTIVE: We sought to develop and validate a simple predictive clinical risk score for SVGD with recurring ischemia after one year post CABG. METHODS AND RESULTS: This was a cross-sectional study and the results were validated using bootstrap resampling on a separate cohort. A nomogram and risk scoring system were developed based on retrospective data from a training cohort of 606 consecutive patients with recurring ischemia >1 year after CABG. Logistic regression model was used to find the predictive factors and to build a nomogram. To assess the generalization, models were validated using bootstrap resampling and an external cross-sectional study of 187 consecutive patients in four other hospitals. In multivariable analysis of the primary cohort, native lesion vessel number, SVG age, recurring ischemia type, very low-density lipoprotein level, and left ventricular end-diastolic diameter were independent predictors. A summary risk score was derived from nomogram, with a cut-off value of 15. In internal and external validation, the C-index was 0.86 and 0.82, indicating good discrimination. The calibration curve for probability of SVGD showed optimal agreement between actual observations and risk score prediction. CONCLUSION: A simple-to-use risk scoring system based on five easily variables was developed and validated to predict the risk of SVGD among patients who recurring ischemia after one year post CABG. This score may be useful for providing patients with individualized estimates of SVGD risk.

Berbamine Hydrochloride inhibits lysosomal acidification by activating Nox2 to potentiate chemotherapy-induced apoptosis via the ROS-MAPK pathway in human lung carcinoma cells.

Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

Fangchinoline inhibits non-small cell lung cancer metastasis by reversing epithelial-mesenchymal transition and suppressing the cytosolic ROS-related Akt-mTOR signaling pathway.

Few drugs alleviate non-small cell lung cancer (NSCLC) metastasis effectively. Small molecular screening demonstrated that fangchinoline (Fan) reversed epithelial-mesenchymal transition (EMT) in NSCLC cells, inhibiting cell invasion and migration. RNA sequencing (RNA-seq) of Fan-treated NSCLC cells revealed that Fan potently quenched the NADP+ metabolic process. Molecular docking analysis revealed that Fan directly and specifically targeted NOX4. NOX4 was associated with poor prognosis in NSCLC in both The Cancer Genome Atlas (TCGA) and Hong Kong cohorts. In mitochondrial DNA-depleted ρ0 NSCLC cells, Fan decreased cytosolic reactive oxygen species (ROS) to inhibit the Akt-mTOR signaling pathway by directly promoting NOX4 degradation. In TCGA and Hong Kong cohorts, NOX4 upregulation acted as a driver event as it positively correlated with metastasis and oxidative stress. Single-cell RNA-seq indicated that NOX4 was overexpressed, especially in cancer cells, cancer stem cells, and endothelial cells. In mice, Fan significantly impeded subcutaneous xenograft formation and reduced metastatic nodule numbers in mouse lung and liver. Drug sensitivity testing demonstrated that Fan suppressed patient-derived organoid growth dose-dependently. Fan is a potent small molecule for alleviating NSCLC metastasis by directly targeting NOX4 and is a potential novel therapeutic agent.

Integrated analysis reveals the pivotal interactions between immune cells in the melanoma tumor microenvironment.

Melanoma is the most lethal type of skin cancer. Despite the breakthroughs in the clinical treatment of melanoma using tumor immunotherapy, many patients do not benefit from these immunotherapies because of multiple immunosuppressive mechanisms. Therefore, there is an urgent need to determine the mechanisms of tumor-immune system interactions and their molecular determinants to improve cancer immunotherapy. In this study, combined analysis of microarray data and single-cell RNA sequencing data revealed the key interactions between immune cells in the melanoma microenvironment. First, differentially expressed genes (DEGs) between normal and malignant tissues were obtained using GEO2R. The DEGs were then subjected to downstream analyses, including enrichment analysis and protein-protein interaction analysis, indicating that these genes were associated with the immune response of melanoma. Then, the GEPIA and TIMER databases were used to verify the differential expression and prognostic significance of hub genes, and the relationship between the hub genes and immune infiltration. In addition, we combined single cell analysis from GSE123139 to identify immune cell types, and validated the expression of the hub genes in these immune cells. Finally, cell-to-cell communication analysis of the proteins encoded by the hub genes and their interactions was performed using CellChat. We found that the CCL5-CCR1, SELPLG-SELL, CXCL10-CXCR3, and CXCL9-CXCR3 pathways might play important roles in the communication between the immune cells in tumor microenvironment. This discovery may reveal the communication basis of immune cells in the tumor microenvironment and provide a new idea for melanoma immunotherapy.

Tubeimoside I-induced lung cancer cell death and the underlying crosstalk between lysosomes and mitochondria.

Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.

Hederagenin potentiated cisplatin- and paclitaxel-mediated cytotoxicity by impairing autophagy in lung cancer cells.

Autophagy inhibition has been demonstrated to increase the efficacy of conventional chemotherapy. In this study, we identified hederagenin, a triterpenoid derived from Hedera helix, as a potent inhibitor of autophagy and then hypothesized that hederagenin might synergize with chemotherapeutic drugs (e.g., cisplatin and paclitaxel) to kill lung cancer cells. Firstly, we observed that hederagenin induced the increased autophagosomes in lung cancer cells concomitantly with the upregulation of LC3-II and p62, which indicated the impairment of autophagic flux. The colocalization assay indicated hederagenin could not block the fusion of lysosomes and autophagosomes, whereas the lysosomal acidification might be inhibited by hederagenin as revealed by the reduced staining of acidity-sensitive reagents (i.e., Lysotracker and acridine orange). The aberrant acidic environment then impaired the function of lysosome, which was evidenced by the decrease of mature cathepsin B and cathepsin D. Lastly, hederagenin, in agree with our hypothesis, promoted pro-apoptotic effect of cisplatin and paclitaxel with the accumulation of reactive oxygen species (ROS); while the synergistic effect could be abolished by the ROS scavenger, N-acetyl-L-cysteine. These data summarily demonstrated hederagenin-induced accumulation of ROS by blocking autophagic flux potentiated the cytotoxicity of cisplatin and paclitaxel in lung cancer cells.

Screening and Identification of Potential Biomarkers for Hepatocellular Carcinoma: An Analysis of TCGA Database and Clinical Validation.

INTRODUCTION: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Up to now, many genes associated with HCC have not yet been identified. In this study, we screened the HCC-related genes through the integrated analysis of the TCGA database, of which the potential biomarkers were also further validated by clinical specimens. The discovery of potential biomarkers for HCC provides more opportunities for diagnostic indicators or gene-targeted therapies. METHODS: Cancer-related genes in The Cancer Genome Atlas (TCGA) HCC database were screened by a random forest (RF) classifier based on the RF algorithm. Proteins encoded by the candidate genes and other associated proteins obtained via protein-protein interaction (PPI) analysis were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The newly identified genes were further validated in the HCC cell lines and clinical tissue specimens by Western blotting, immunofluorescence, and immunohistochemistry (IHC). Survival analysis verified the clinical value of genes. RESULTS: Ten genes with the best feature importance in the RF classifier were screened as candidate genes. By comprehensive analysis of PPI, GO and KEGG, these genes were confirmed to be closely related to HCC tumors. Representative NOX4 and FLVCR1 were selected for further validation by biochemical analysis which showed upregulation in both cancer cell lines and clinical tumor tissues. High expression of NOX4 or FLVCR1 in cancer cells predicts low survival. CONCLUSION: Herein, we report that NOX4 and FLVCR1 are promising biomarkers for HCC that may be used as diagnostic indicators or therapeutic targets.

Synergistic inhibitory effect of resveratrol and TK/GCV therapy on melanoma cells.

PURPOSE: To investigate the synergistic effect of resveratrol on the bystander effect of TK/GCV suicide gene system in melanoma cells. METHODS: The effect of resveratrol on the growth of B16 cells and the synergistic effect of resveratrol with or without GCV were detected by MTT assay and high content screening assay. The effect of resveratrol on GJIC function was detected by flow cytometry combined with fluorescence tracer and fluorescence microscope, and the expression of gap junction protein was detected by western blotting. Synergistic killing effect of resveratrol plus TK/GCV was tested in vivo using transplanted melanoma model. RESULTS: In vitro, resveratrol can enhanced GJ function and upregulated Cx32 and Cx43 protein expression in B16 cells. Resveratrol synergized with GCV to kill mixed B16 melanoma cells (20% TK+ cells and 80% TK- cells) and to improve apoptosis rate of TK- cells (the bystander effect of TK system), and the synergistic action was reversed by the GJ inhibitor AGA. In vivo, when B16 cells were mixed with 30% TK+ B16 cells, significantly reduced tumor weight and volume were observed after combinational treatment with resveratrol plus GCV as compared with GCV or resveratrol treatment alone. CONCLUSIONS: Resveratrol could synergistically enhance the killing effect of TK/GCV suicide gene system in melanoma B16 cells and transplanted melanoma. It might be a promising adjuvant of TK/GCV therapy.

Prediction of Major Adverse Cardiovascular Events and Slow/No-Reflow by Virtual Histology Imaging After Percutaneous Interventions on Saphenous Vein Grafts.

Saphenous vein grafts disease (SVGD) is a common complication after coronary artery bypass graft (CABG) and usually treated by percutaneous coronary intervention (PCI). In this prospective cohort study, we performed virtual histology-intravascular ultrasound to investigate whether plaque composition and morphological characteristics were associated with post-PCI major adverse cardiac events (MACEs) and slow/no-reflow in patients with SVGD. Patients (n = 90) were studied (76.7% men, mean age 64.9 ± 8.2 years and mean duration of SVG 8.0 ± 3.6 years). There were 77.8% lesions with a plaque burden of at least 70%; 18 MACE incidences accumulated in 14 patients over 12 months post-PCI and slow/no-reflow was observed in 12 patients. On adjusted multivariate analysis, lesion length (hazard ratio [HR] = 1.05; 95% confidence interval [CI]: 1.01-1.08]); age of CABG (HR = 1.51 [95% CI: 1.11-2.05], and absolute necrotic core (NC) area (HR = 8.04 [95% CI: 1.86-34.73]) were independently associated with MACEs. Factors independently associated with slow/no-reflow post-PCI were preprocedure systolic blood pressure (odds ratio [OR] = 0.98; 95% CI: 0.96-0.99) and absolute NC area (OR = 2.47 (95% CI: 1.14-5.36). A cutoff value of absolute NC area at ≥1.1 mm2 may serve as a significant risk predictor for no-reflow after SVG-PCI. Factors associated with MACEs and the slow/no-reflow phenomenon following PCI of the SVG can be used in risk assessment of SVG.

N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells.

The main mechanistic function of most chemotherapeutic drugs is mediated by inducing mitochondria-dependent apoptosis. Tumor cells usually respond to upregulate autophagy to eliminate impaired mitochondria for survival. Hypothetically, inhibiting autophagy might promote mitochondria-dependent apoptosis, thus enhancing the efficacy of chemotherapeutic therapies. We previously identified N-methylparoxetine (NMP) as an inducer of mitochondrial fragmentation with subsequent apoptosis in non-small cell lung cancer (NSCLC) cells. We discovered that ROS was accumulated in NMP-treated NSCLC cells, followed by c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) activation. This was reversed by the application of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), leading to a reduction in apoptosis. Our data suggested that NMP induced apoptosis in NSCLC cells by activating mitogen-activated protein kinase (MAPK) pathway. We further speculated that the remarkable increase of ROS in NMP-treated NSCLC cells might result from an inhibition of autophagy. Our current data confirmed that NMP blocked autophagy flux at late stage wherein lysosomal acidification was inhibited. Taken together, this study demonstrated that NMP could exert dual apoptotic functions-mitochondria impairment and, concomitantly, autophagy inhibition. NMP-related excessive ROS accumulation induced apoptosis by activating the MAPK pathway in NSCLC cells.

Dioscin Inhibits the Invasion and Migration of Hepatocellular Carcinoma HepG2 Cells by Reversing TGF-ß1-Induced Epithelial-Mesenchymal Transition.

Dioscin is a natural steroidal saponin that can be isolated from Chinese medicine, such as Dioscoreae rhizoma. It has wild range of pharmacological activities such as hepatoprotection, a lipid-lowering effect, and anti-inflammation. Recently, mounting studies reported the anticancer effect of dioscin on a variety of tumor cells. However, the potential effect of dioscin on the epithelial-mesenchymal transition (EMT) of HepG2 cells is unclear. In the present study, dioscin was identified to inhibit transforming growth factor-ß1 (TGF-ß1) and induced invasive and migratory behavior of HepG2 cells. Consistently, the expression of the epithelial marker E-cadherin and gap junction proteins increased following dioscin treatment, while mesenchymal markers decreased, including N-cadherin, Vimentin, Snail, and Slug. Furthermore, we discovered that TGF-ß1 induces phosphorylation of JNK, p38, and Erk, whereas the activation of these kinases was reversed by dioscin treatment in a dose-dependent manner. With the addition of Asiatic acid, a p38 activator, the inhibitory effect of dioscin on EMT was reversed. Taken together, these data indicated that dioscin inhibits EMT in HepG2 cells, which is mediated in large part by inhibition of the p38-MAPK signaling.

Antitumor effects of circ-EPHB4 in hepatocellular carcinoma via inhibition of HIF-1α.

The protein EPHB4 plays a vital role in various tumor types. However, few studies into the function of circ-EPHB4 (hsa_circ_0001730) in tumors have been conducted. This study aimed to investigate the functions of circ-EPHB4 and the underlying mechanism of circ-EPHB4 in regulating hepatocellular carcinoma (HCC). The expression of circ-EPHB4 was found to be downregulated in HCC tumor tissues, whereas circ-EPHB4 overexpression suppressed cell viability, induced apoptosis, and inhibited cell migration and invasion in Huh7 and HepG2 cells. circ-EPHB4 levels were negatively correlated with tumor weight, size, and metastasis foci in nude mouse models, suggesting circ-EPHB4 inhibits tumorigenesis, tumor development, and metastasis. In addition, HIF-1α and PI3K-AKT pathways were markedly affected by circ-EPHB4 overexpression. HIF-1α could potentially be the target of circ-EPHB4. By overexpressing both HIF-1α and circ-EPHB4, the antitumor effect of circ-EPHB4 should be most probably correlated with HIF-1α. In conclusion, circ-EPHB4 is a tumor inhibitor in HCC and functions by inhibiting HIF-1α expression.

Synergistic effect of resveratrol and HSV-TK/GCV therapy on murine hepatoma cells.

Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.

Blockage of Autophagic Flux and Induction of Mitochondria Fragmentation by Paroxetine Hydrochloride in Lung Cancer Cells Promotes Apoptosis via the ROS-MAPK Pathway.

Cancer cells are characterized by malignant proliferation and aberrant metabolism and are thereby liable to the depletion of nutrients and accumulation of metabolic waste. To maintain cellular homeostasis, cancer cells are prone to upregulating the canonical autophagy pathway. Here, we identified paroxetine hydrochloride (Paxil) as a late autophagy inhibitor and investigated its killing effect on lung cancer cells and with a xenograft mouse model in vivo. Upregulated LC3-II and p62 expression indicated that Paxil inhibited autophagy. Acid-sensitive dyes (e.g., LysoTracker and AO staining) indicated reduced lysosomal acidity following Paxil treatment; consequently, the maturation of the pH-dependent hydroxylases (e.g., cathepsin B and D) substantially declined. Paxil also induced the fragmentation of mitochondria and further intensified ROS overproduction. Since the autophagy pathway was blocked, ROS rapidly accumulated, which activated JNK and p38 kinase. Such activity promoted the localization of Bax, which led to increased mitochondrial outer membrane permeability. The release of Cytochrome c with the loss of the membrane potential triggered a caspase cascade, ultimately leading to apoptosis. In contrast, the clearance of ROS by its scavenger, NAC, rescued Paxil-induced apoptosis accompanied by reduced p38 and JNK activation. Thus, Paxil blocked the autophagic flux and induced the mitochondria-dependent apoptosis via the ROS-MAPK pathway.

Relationship between plaque composition by virtual histology intravascular ultrasound and clinical outcomes after percutaneous coronary intervention in saphenous vein graft disease patients: study protocol of a prospective cohort study.

BACKGROUND: Plaque composition and morphologic characteristics identified by virtual histology intravascular ultrasound (VH-IVUS) can determine plaques at increased risk of clinical events following percutaneous coronary intervention (PCI) among coronary artery disease (CAD) patients. However, there have been few studies to investigate the relationship between plaque composition of saphenous vein graft (SVG) by VH-IVUS and clinical outcomes in patients with saphenous vein graft disease (SVGD) undergoing PCI. The purpose of this study is to determine whether plaque components and characteristics by VH-IVUS can predict major adverse cardiac events (MACEs) among SVGD patients undergoing PCI. METHODS/DESIGN: This is a prospective cohort study conducted in Tianjin Chest Hospital, China. Participants with SVGD referred for PCI will be invited to participate in this study, and will be followed up at 1, 6, 12, 24 and 36 months post-PCI to assess clinical outcomes. The planned sample size is 175 subjects. We will recruit subjects with SVGD scheduled to receive PCI, aged 18-80 years, with a history of previous coronary artery bypass graft (CABG) surgery more than 1 year ago, and willing to participate in the study and sign informed consent. The composite primary study endpoint is the incidence of MACEs after PCI for SVGD, including death from cardiac causes, non-fatal myocardial infarction, unplanned target lesion revascularization (TLR) and target vessel revascularization (TVR). The primary outcome analysis will be presented as Kaplan-Meier estimates and the primary outcome analysis will be carried out using a Cox proportional hazards regression model. DISCUSSION: Once the predictive values of plaque components and characteristics by VH-IVUS on subsequent clinical outcomes are determined among SVGD patients undergoing PCI, an innovative prediction tool of clinical outcomes for SVGD patients undergoing PCI will be created, which may lead to the development of new methods of risk stratification and intervention guidance. TRIAL REGISTRATION: The study is registered to ClinicalTrials.gov (NCT03175952).

The Novel Autophagy Inhibitor Alpha-Hederin Promoted Paclitaxel Cytotoxicity by Increasing Reactive Oxygen Species Accumulation in Non-Small Cell Lung Cancer Cells.

Chemoresistance is a major limiting factor that impairs the outcome of non-small cell lung cancer (NSCLC) chemotherapy. Paclitaxel (Tax) induces protective autophagy in NSCLC cells, leading to the development of drug resistance. We recently identified a new autophagy inhibitor (alpha-hederin) and hypothesized that it may promote the killing effect of Tax on NSCLC cells. We found that alpha-hederin (α-Hed) could block late autophagic flux in NSCLC cells by altering lysosomal pH and inhibiting lysosomal cathepsin D maturation. Combination treatment of α-Hed and Tax synergistically reduced NSCLC cell proliferation and increased NSCLC cell apoptosis compared with treatment with α-Hed or Tax alone. Furthermore, α-Hed plus Tax enhanced the accumulation of intracellular reactive oxygen species (ROS) in NSCLC cells, while the ROS inhibitor N-acetylcysteine reversed the inhibitory effect of the combination treatment. Our findings suggest that α-Hed can increase the killing effect of Tax on NSCLC cells by promoting ROS accumulation, and that combining α-Hed with classical Tax represents a novel strategy for treating NSCLC.

Association between proprotein convertase subtilisin/kexin type 9 and late saphenous vein graft disease after coronary artery bypass grafting: a cross-sectional study.

OBJECTIVE: The study aims to explore the association between serum proprotein convertase subtilisin/kexin type 9 (PCSK9) level and saphenous vein grafts disease (SVGD) after coronary artery bypass grafting (CABG). DESIGN: A cross-sectional study. SETTING: A secondary hospital in Tianjin City, China. PARTICIPANTS: A total of 231 participants were included in the study. Inclusion criteria were as follows: age ≥18 years, previous CABG surgery at least 12 months ago, at least one SVG for bypass during CABG, abnormal non-invasive test results or recurrent stable angina pectoris by coronary angiography indications, and willing to participate and sign informed consent. Participants with any of the following were excluded from the study: congenital valvular disease, decompensated heart failure, anaemia defined as a haemoglobin level of <12 g/dL in women or <13 g/dL in men, malignant neoplasms, renal failure, severe hepatic disease, thyroid disease, acute or chronic inflammatory disease and chronic obstructive lung disease. PRIMARY OUTCOME MEASURE: SVGD was defined as at least one SVG with significant stenosis (≥50%). Circulating PCSK9 levels were measured using commercial ELISA kits according to the manufacturer's instructions. RESULTS: The mean PCSK9 level in the SVGD group was significantly higher than that in the patent group (275.2±38.6 vs 249.3±37.7, p<0.01). The multivariate logistic regression model revealed a significant association between serum PCSK9 and SVGD (OR 2.08, 95% CI 1.46-2.95) per 1 SD increase in serum PCSK9. CONCLUSIONS: The present study is the first to identify an independent association between PCSK9 and late SVGD after adjustment for established cardiovascular risk factors. A multicentre prospective cohort study with large sample size should be conducted in the future to further research this relationship.