Zhendan He

Induction of apoptosis in human leukemia cells through an intrinsic pathway by cathachunine, a unique alkaloid isolated from Catharanthus roseus

BACKGROUNDnCatharanthus roseus (L.) G. Don consists of a range of dimeric indole alkaloids with significant antitumor activities. These alkaloids have been found to possess apoptosis-inducing activity against tumor cells in vitro and in vivo mediated by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, in which DNA damage and mitochondrial dysfunction play important roles. In this study, a unique bisindole alkaloid named cathachunine, along with five known dimeric indole alkaloids, was obtained from C. roseus and investigated in vitro.nnnPURPOSEnThe aim of this study was to investigate the antitumor activity of isolated alkaloids and the mechanism through which cathachunine exerts its antitumor effect.nnnSTUDY DESIGN AND METHODSnCell growth inhibition was assessed by WST-1 and lactate dehydrogenase (LDH) assays in HL60, K562 leukemia cells and EA.hy926 umbilical vein cells. Induction of apoptosis in HL60 cells was confirmed by observation of nuclear morphology, a caspase-3 activity assay and annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) double staining. The intrinsic apoptotic pathway induced by cathachunine was evidenced by B-cell lymphoma 2/Bcl-2-associated X protein (Bcl-2/Bax) dysregulation, loss of mitochondrial membrane potential, translocation of cytochrome c, and cleavage of caspase-3 and poly-ADP ribose polymerase (PARP). Reactive oxygen species (ROS) production after cathachunine treatment was determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Cell cycle arrest of the S phase was also observed in HL60 cells after cathachunine treatment.nnnRESULTSnThe WST-1 and LDH assays showed that Catharanthus alkaloids were cytotoxic toward human leukemia cells to a greater extent than toward normal human endothelial cells, and the anti-proliferation and pro-apoptosis abilities of cathachunine were much more potent than other previously reported alkaloids. The induction of apoptosis by cathachunine occurred through an ROS-dependent mitochondria-mediated intrinsic pathway rather than an extrinsic pathway, and was regulated by the Bcl-2 protein family.nnnCONCLUSIONnAn unprecedented bisindole alkaloid cathachunine which lost C-18 and C-19 was isolated from C. roseus. It exerted a potent antitumor effect toward human leukemia cells through the induction of apoptosis via an intrinsic pathway. Thus, this study provides evidence for a new lead compound from a natural source for anti-cancer investigations.

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

ABSTRACT Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.

Anti-influenza virus effects of crude phenylethanoid glycosides isolated from ligustrum purpurascens via inducing endogenous interferon-γ

ETHNOPHARMACOLOGICAL RELEVANCEnLigustrum purpurascens Y.C. Yang (Oleaceae) is traditionally recorded as Ku Ding Cha, a kind of functional tea in southern China for about two thousand years, which has been reported with sore throat alleviating and pathogenic heat expelling effects. However, there are no scientific studies demonstrating its antiviral activity.nnnTHE AIM OF THE STUDYnThis study is aimed at investigating the anti-influenza virus effects of phenylethanoid glycosides isolated from L. purpurascens (LPG) as well as its corresponding mechanisms.nnnMATERIALS AND METHODSnIn vitro, hemagglutination assay was employed to detect the influenza virus titer; In vivo, C57BL/6J mice were given oral administration of LPG (100mg/kg, 300mg/kg, 900mg/kg) or ribavirin (100mg/kg) once daily for 5 successive days. Meanwhile, on the second day, mice were infected intranasally (i.n.) with A/FM/1/47 H1N1 virus. Mice survival rate and other clinical index were monitored for 15 days. Infected mice were sacrificed to measure the lung lesion and stained with hematoxylin-eosin. Flow cytometry analyses spleen lymphocytes and interferon-γ (IFN-γ) level. The IFN-γ knockout mice (IFN-γ(-/-) mice, C57BL/6J) which had been verified lacking IFN-γ through Western Blot, were applied in the death-protection test to identify the role of IFN-γ played in LPG antiviral effect.nnnRESULTSnIn vitro, LPG at 0.5mg/ml inhibited Influenza A Virus H1N1 type (H1N1) infection of MDCK cells. In vivo, LPG at 300 and 900mg/kg significantly decreased the mouse lung index (p<0.05), alleviated influenza-induced lethality and clinical symptoms, and therefore enhanced mouse survival (p<0.05). More detailed experiments demonstrated that antiviral cytokine IFN-γ was involved in the antiviral effect of LPG. Flow cytometric analysis revealed that LPG (900mg/kg) significantly induced secretion of IFN-γ by splenic CD4(+) and CD8(+) cells (p<0.05). Moreover, LPG (900mg/kg) protected wild-type C57BL/6J mice from H1N1 injury, whereas LPG-mediated survival protection disappeared in IFN-γ(-/-) mice.nnnCONCLUSIONnThese results suggest that up-regulating endogenous IFN-γ by LPG may represent a novel therapeutic approach for H1N1 infection.

Crude Saponins of Panax notoginseng Have Neuroprotective Effects To Inhibit Palmitate-Triggered Endoplasmic Reticulum Stress-Associated Apoptosis and Loss of Postsynaptic Proteins in Staurosporine Differentiated RGC-5 Retinal Ganglion Cells

Increased apoptosis of retinal ganglion cells (RGCs) contributes to the gradual loss of retinal neurons at the early phase of diabetic retinopathy (DR). There is an urgent need to search for drugs with neuroprotective effects against apoptosis of RGCs for the early treatment of DR. This study aimed to investigate the neuroprotective effects of saponins extracted from Panax notoginseng, a traditional Chinese medicine, on apoptosis of RGCs stimulated by palmitate, a metabolic factor for the development of diabetes and its complications, and to explore the potential molecular mechanism. We showed that crude saponins of P. notoginseng (CSPN) inhibited the increased apoptosis and loss of postsynaptic protein PSD-95 by palmitate in staurosporine-differentiated RGC-5 cells. Moreover, CSPN suppressed palmitate-induced reactive oxygen species generation and endoplasmic reticulum stress-associated eIF2α/ATF4/CHOP and caspase 12 pathways. Thus, our findings address the potential therapeutic significance of CSPN for the early stage of DR.

Dual-index evaluation of character changes in Panax ginseng C. A. Mey stored in different conditions.

Panax ginseng C. A. Mey has been used as a traditional medicine and functional food in Asia for thousands of years for its improvement of human immunity and metabolism and its antitumor and antifatigue activities. This study reports the impact of storage conditions and storage period on the quality of P. ginseng. The contents of four major ginsenosides in P. ginseng and phosphorylation activities of Akt of ginseng extracts were affected by both storage conditions and storage period. In contrast, the ATP generation capacity of ginseng extracts was affected by storage conditions, but not by storage period. The results showed that the quality of P. ginseng could be well maintained at a relative humidity between 70% and 90%, and dry conditions might decrease the quality of P. ginseng. Through dual-index evaluation, the present study extended our knowledge on the changes of ginsenosides and bioactivities in P. ginseng with respect to different storage conditions and storage periods.

Gypenoside L inhibits autophagic flux and induces cell death in human esophageal cancer cells through endoplasm reticulum stress-mediated Ca 2+ release

Esophageal cancer is one of the leading cause of cancer mortality in the world. Due to the increased drug and radiation tolerance, it is urgent to develop novel anticancer agent that triggers nonapoptotic cell death to compensate for apoptosis resistance. In this study, we show that treatment with gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, induced nonapoptotic, lysosome-associated cell death in human esophageal cancer cells. Gyp-L-induced cell death was associated with lysosomal swelling and autophagic flux inhibition. Mechanistic investigations revealed that through increasing the levels of intracellular reactive oxygen species (ROS), Gyp-L triggered protein ubiquitination and endoplasm reticulum (ER) stress response, leading to Ca2+ release from ER inositol trisphosphate receptor (IP3R)-operated stores and finally cell death. Interestingly, there existed a reciprocal positive-regulatory loop between Ca2+ release and ER stress in response to Gyp-L. In addition, protein synthesis was critical for Gyp-L-mediated ER stress and cell death. Taken together, this work suggested a novel therapeutic option by Gyp-L through the induction of an unconventional ROS-ER-Ca2+-mediated cell death in human esophageal cancer.

Cellular defence or viral assist: the dilemma of HDAC6

Histone deacetylase 6 (HDAC6) is a unique cytoplasmic deacetylase that regulates various important biological processes by preventing protein aggregation and deacetylating different non-histone substrates including tubulin, heat shock protein 90, cortactin, retinoic acid inducible gene I and β-catenin. Growing evidence has indicated a dual role for HDAC6 in viral infection and pathogenesis: HDAC6 may represent a host defence mechanism against viral infection by modulating microtubule acetylation, triggering antiviral immune response and stimulating protective autophagy, or it may be hijacked by the virus to enhance proinflammatory response. In this review, we will highlight current data illustrating the complexity and importance of HDAC6 in viral pathogenesis. We will summarize the structure and functional specificity of HDAC6, and its deacetylase- and ubiquitin-dependent activity in key cellular events in response to virus infection. We will also discuss how HDAC6 exerts its direct or indirect histone modification ability in viral lytic-latency switch.

Gypenoside L, Isolated from Gynostemma pentaphyllum, Induces Cytoplasmic Vacuolation Death in Hepatocellular Carcinoma Cells through Reactive-Oxygen-Species-Mediated Unfolded Protein Response.

Exploring novel anticancer agents that can trigger non-apoptotic or non-autophagic cell death is urgent for cancer treatment. In this study, we screened and identified an unexplored anticancer activity of gypenoside L (Gyp-L) isolated from Gynostemma pentaphyllum. We showed that treatment with Gyp-L induces non-apoptotic and non-autophagic cytoplasmic vacuolation death in human hepatocellular carcinoma (HCC) cells. Mechanically, Gyp-L initially increased the intracellular reactive oxygen species (ROS) levels, which, in turn, triggered protein ubiquitination and unfolded protein response (UPR), resulting in Ca(2+) release from endoplasm reticulum (ER) inositol trisphosphate receptor (IP3R)-operated stores and finally cytoplasmic vacuolation and cell death. Interruption of the ROS-ER-Ca(2+) signaling pathway by chemical inhibitors significantly prevented Gyp-L-induced vacuole formation and cell death. In addition, Gyp-L-induced ER stress and vacuolation death required new protein synthesis. Overall, our works provide strong evidence for the anti-HCC activity of Gyp-L and suggest a novel therapeutic option by Gyp-L through the induction of a unconventional ROS-ER-Ca(2+)-mediated cytoplasmic vacuolation death in human HCC.

SMG-1 inhibition by miR-192/-215 causes epithelial-mesenchymal transition in gastric carcinogenesis via activation of Wnt signaling

SMG‐1,a member of the phosphoinositide kinase‐like kinase family, functioned as a tumor suppressor gene. However, the role of SMG‐1 in GC remain uncharacterized. In this study, regulation of SMG‐1 by miR‐192 and‐215, along with the biological effects of this modulation, were studied in GC. We used gene microarrays to screening and luciferase reporter assays were to verify the potential targets of miR‐192 and‐215. Tissue microarrays analyses were applied to measure the levels of SMG‐1 in GC tissues. Western blot assays were used to assess the signaling pathway of SMG‐1 regulated by miR‐192 and‐215 in GC. SMG‐1 was significantly downregulated in GC tissues.The proliferative and invasive properties of GC cells were decreased by inhibition of miR‐192 and‐215, whereas an SMG‐1siRNA rescued the inhibitory effects. Finally, SMG‐1 inhibition by miR‐192 and‐215 primed Wnt signaling and induced EMT. Wnt signaling pathway proteins were decreased markedly by inhibitors of miR‐192 and‐215, while SMG‐1 siRNA reversed the inhibition apparently. Meanwhile, miR‐192 and‐215 inhitibtors increased E‐cadherin expression and decreased N‐cadherin and cotransfection of SMG‐1 siRNA reversed these effects. In summary, these findings illustrate that SMG‐1 is suppressed by miR‐192 and‐215 and functions as a tumor suppressor in GC by inactivating Wnt signaling and suppressing EMT.

Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton

Abstract Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host–pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies.