Dengue virus (DENV) utilizes the endoplasmic reticulum (ER) for replication and assembling. Accumulation of unfolded proteins in the ER lumen leads to ER stress and unfolded protein response (UPR). ...Three branches of UPRs temporally modulated DENV infection. Moreover, ER stress can also induce autophagy. DENV infection induces autophagy which plays a promotive role in viral replication has been reported. However, the role of ER stress in DENV-induced autophagy, viral titer, and pathogenesis remain unclear. Here, we reveal that ER stress and its downstream UPRs are indispensable for DENV-induced autophagy in various human cells. We demonstrate that PERK-eIF2α and IRE1α-JNK signaling pathways increased autophagy and viral load after DENV infection. However, ATF6-related pathway showed no effect on autophagy and viral replication. IRE1α-JNK downstream molecule Bcl-2 was phosphorylated by activated JNK and dissociated from Beclin 1, which playing a critical role in autophagy activation. These findings were confirmed as decreased viral titer, attenuated disease symptoms, and prolonged survival rate in the presence of JNK inhibitor in vivo. In summary, we are the first to reveal that DENV2-induced ER stress increases autophagy activity, DENV replication, and pathogenesis through two UPR signaling pathways both in vitro and in vivo.
The global incidence of thyroid cancer, one of the most common endocrine malignancies, is especially high among women. Although most patients with thyroid cancers exhibit a good prognosis with ...standard treatment, there are no effective therapies for patients with anaplastic thyroid cancers or cancers that have reached an advanced or recurrent level. Therefore, it is important to develop highly effective compounds for treating such patients. Aloperine, a natural compound isolated from
, has been reported to possess antioxidant, anti-inflammatory, anti-neuronal injury, anti-renal injury, antitumor, anti-allergic, and antiviral properties. In this study, we show that aloperine can inhibit cell growth in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers. Moreover, it could suppress in vitro tumorigenesis and promote cellular apoptosis. Further analysis demonstrated the involvement of caspase-dependent apoptosis, including intrinsic and/or extrinsic pathways, in aloperine-induced cellular apoptosis. However, cell cycle regulation was not detected with aloperine treatment. This study suggests the potential therapeutic use of aloperine in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers.
Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms ...of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down‐regulation of Cdk2 and Cdk4 and the up‐regulation of cell cycle suppressors, p21 and p27. In addition, the caspase‐dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3‐MA or bafilomycin, potentiated the honokiol‐mediated anti‐OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5‐FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5‐FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC‐xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.
Lung cancer is one of the most common cancers and the leading cause of death in humans worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases and is often ...diagnosed at a late stage. Among patients with NSCLC, 50% die within 1 year after diagnosis. Even with clinical intervention, the 5-year survival rate is only approximately 20%. Therefore, the development of an advanced therapeutic strategy or novel agent is urgently required for treating NSCLC. Berberine exerts therapeutic activity toward NSCLC; therefore, its activity as an antitumor agent needs to be explored further. In this study, three terpenylated-bromide derivatives of berberrubine were synthesized and their anti-NSCLC activities were evaluated. Each derivative had higher anti-NSCLCs activity than berberrubine and berberine. Among them, 9-O-gernylberberrubine bromide (B4) and 9-O-farnesylberberrubine bromide (B5) showed greater growth inhibition, cell-cycle regulation, in vitro tumorigenesis suppression, and tumor migration reduction. In addition, some degree of apoptosis and autophagic flux blocking was noted in the cells under B4 and B5 treatments. Our study demonstrates that the berberrubine derivatives, B4 and B5, exhibit impressive anti-NSCLC activities and have potential for use as chemotherapeutic agents against NSCLC.
Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy‐mediated exocytosis of IL‐1β, ...insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β‐cell “Min‐6” and human lung cancer cell “CL1‐5‐Q89L” with high secretory autophagy tendency by LC–MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy‐related vesicle trafficking. We used CL1‐5‐Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy‐related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.
Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that ...enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.
Lung cancer is the leading cause of death in the world, and the most common type of lung cancer is non-small-cell lung cancer (NSCLC), accounting for 85% of lung cancer. Patients with NSCLC, when ...detected, are mostly in a metastatic stage, and over half of patients diagnosed with NSCLC die within one year after diagnosis; the 5-year survival rate is 24%. However, in patients with metastatic NSCLC, the 5-year survival rate is 6%. Therefore, development of a new therapeutic agent or strategy is urgent for NSCLCs. Berberine has been illustrated to be a therapeutic agent of NSCLC. In the present study, we synthesized six derivatives of berberine, and the anti-NSCLC activity of these agents was examined. Some of them exert increasing proliferation inhibition comparing with berberine. Further studies demonstrated that two of the most effective agents, 9-
-decylberberrubine bromide (B6) and 9-
-dodecylberberrubine bromide (B7), performed cell cycle regulation, in-vitro tumorigenesis inhibition and autophagic flux blocking, but not induction of cellular apoptosis in NSCLC cells. Moreover, B6 and B7 were determined to be green fluorescent and could be penetrated and localized in cellular mitochondria. Herein, B6 and B7, the berberine derivatives we synthesized, revealed better anti-NSCLC activity with berberine and may be used as therapeutic candidates for the treatment of NSCLCs.
Thyroid cancer (TC) is the most common endocrine malignancy. Recently, the global incidence of TC has increased rapidly. Differentiated thyroid cancer includes papillary thyroid carcinoma (PTC) and ...follicular thyroid carcinoma (FTC), which are the most common types of TC. Although PTCs and FTCs exert good prognoses and high survival rates, FTCs tend to be more aggressive than PTCs. There is an urgent need to improve patient outcomes by developing effective therapeutic agents for FTCs. Piperlongumine exerts anti-cancer effects in various human carcinomas, including human anaplastic TCs and PTCs. However, the anti-cancer effects of piperlongumine in FTCs and the underlying mechanisms are yet to be elucidated. Therefore, in the present study, we evaluated the effect of piperlongumine on cell proliferation, cell cycle, apoptosis, and autophagy in FTC cells with flowcytometry and Western blot. We observed that piperlongumine caused growth inhibition, cell cycle arrest, apoptosis induction, and autophagy elevation in FTC cells. Activities of reactive oxygen species and the downstream PI3K/Akt pathway were the underlying mechanisms involved in piperlongumine mediated anti-FTC effects. Advancements in our understanding of the effects of piperlongumine in FTC hold promise for the development of novel therapeutic strategies.
Thyroid cancer (TC) is the most common endocrine malignancy, and its global incidence has steadily increased over the past 15 years. TC is broadly divided into well-differentiated, poorly ...differentiated, and undifferentiated types, depending on the histological and clinical parameters. Thus far, there are no effective treatments for undifferentiated thyroid cancers or advanced and recurrent cancer. Therefore, the development of an effective therapeutic is urgently needed for such patients. Piperlongumine (PL) is a naturally occurring small molecule derived from long pepper; it is selectively toxic to cancer cells by generating reactive oxygen species (ROS). In this study, we demonstrate the potential anticancer activity of PL in four TC cell lines. For this purpose, we cultured TC cell lines and analyzed the following parameters: Cell viability, colony formation, cell cycle, apoptosis, and cellular ROS induction. PL modulated the cell cycle, induced apoptosis, and suppressed tumorigenesis in TC cell lines in a dose- and time-dependent manner through ROS induction. Meanwhile, an intrinsic caspase-dependent apoptosis pathway was observed in the TC cells under PL treatment. The activation of Erk and the suppression of the Akt/mTOR pathways through ROS induction were seen in cells treated with PL. PL-mediated apoptosis in TC cells was through the ROS-Akt pathway. Finally, the anticancer effect and safety of PL were also demonstrated in vivo. Our findings indicate that PL exhibits antitumor activity and has the potential for use as a chemotherapeutic agent against TC. This is the first study to show the sensitivity of TC cell lines to PL.
COVID-19 is threatening human health worldwide but no effective treatment currently exists for this disease. Current therapeutic strategies focus on the inhibition of viral replication or using ...anti-inflammatory/immunomodulatory compounds to improve host immunity, but not both. Traditional Chinese medicine (TCM) compounds could be promising candidates due to their safety and minimal toxicity. In this study, we have developed a novel
bioinformatics workflow that integrates multiple databases to predict the use of honeysuckle (
) and Huangqi (
) as potential anti-SARS-CoV-2 agents. Using extracts from honeysuckle and Huangqi, these two herbs upregulated a group of microRNAs including
,
, and
, which are critical to reduce the pathogenesis of SARS-CoV-2. Moreover, these herbs suppressed pro-inflammatory cytokines including IL-6 or TNF-α, which were both identified in the cytokine storm of acute respiratory distress syndrome, a major cause of COVID-19 death. Furthermore, both herbs partially inhibited the fusion of SARS-CoV-2 spike protein-transfected BHK-21 cells with the human lung cancer cell line Calu-3 that was expressing ACE2 receptors. These herbs inhibited SARS-CoV-2 M
activity, thereby alleviating viral entry as well as replication. In conclusion, our findings demonstrate that honeysuckle and Huangqi have the potential to be used as an inhibitor of SARS-CoV-2 virus entry that warrants further
analysis and functional assessment of miRNAs to confirm their clinical importance. This fast-screening platform can also be applied to other drug discovery studies for other infectious diseases.
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