FATTY ACID DESATURASE4 enhances plant RNA virus replication and undergoes host vacuolar ATPase-mediated degradation.

Emerging evidence indicates that fatty acid (FA) metabolic pathways regulate host immunity to vertebrate viruses. However, information on FA signaling in plant virus infection remains elusive. In this study, we demonstrate the importance of fatty acid desaturase (FAD), an enzyme that catalyzes the rate-limiting step in the conversion of saturated FAs into unsaturated FAs, during infection by a plant RNA virus. We previously found that the rare Kua-ubiquitin-conjugating enzyme (Kua-UEV1) fusion protein FAD4 from Nicotiana benthamiana (NbFAD4) was downregulated upon turnip mosaic virus (TuMV) infection. We now demonstrate that NbFAD4 is unstable and is degraded as TuMV infection progresses. NbFAD4 is required for TuMV replication, as it interacts with TuMV replication protein 6K2 and colocalizes with viral replication complexes. Moreover, NbFAD4 overexpression dampened the accumulation of immunity-related phytohormones and FA metabolites, and its catalytic activity appears to be crucial for TuMV infection. Finally, a yeast 2-hybrid library screen identified the vacuolar H+-ATPase component ATP6V0C as involved in NbFAD4 degradation and further suppression of TuMV infection. This study reveals the intricate role of FAD4 in plant virus infection, and sheds light on a new mechanism by which a V-ATPase is involved in plant antiviral defense.

Long Noncoding RNA AROD Inhibits Host Antiviral Innate Immunity via the miR-324-5p-CUEDC2 Axis.

Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs that are involved in multiple biological processes. Here, we report a mechanism through which the lnc-AROD-miR-324-5p-CUEDC2 axis regulates the host innate immune response, using influenza A virus (IAV) as a model. We identified that host lnc-AROD without protein-coding capability is composed of 975 nucleotides. Moreover, lnc-AROD inhibited interferon-ß expression, as well as interferon-stimulated genes ISG15 and MxA. Furthermore, in vivo assays confirmed that lnc-AROD overexpression increased flu virus pathogenicity and mortality in mice. Mechanistically, lnc-AROD interacted with miR-324-5p, leading to decreased binding of miR-324-5p to CUEDC2. Collectively, our findings demonstrated that lnc-AROD is a critical regulator of the host antiviral response via the miR-324-5p-CUEDC2 axis, and lnc-AROD functions as competing endogenous RNA. Our results also provided evidence that lnc-AROD serves as an inhibitor of the antiviral immune response and may represent a potential drug target. IMPORTANCE lnc-AROD is a potential diagnostic and discriminative biomarker for different cancers. However, so far the mechanisms of lnc-AROD regulating virus replication are not well understood. In this study, we identified that lnc-AROD is downregulated during RNA virus infection. We demonstrated that lnc-AROD enhanced CUEDC2 expression, which in turn inhibited innate immunity and favored IAV replication. Our studies indicated that lnc-AROD functions as a competing endogenous RNA that binds miR-324-5p and reduces its inhibitory effect on CUEDC2. Taken together, our findings reveal that lnc-AROD plays an important role during the host antiviral immune response.

PKC is an indispensable factor in promoting environmental toxin chromium-mediated transformation and drug resistance.

Hexavalent chromium [Cr(VI)] pollution is a serious environmental problem, due to not only its toxicity but also carcinogenesis. Although studies reveal several features of Cr(VI)-induced carcinogenesis, the underlying mechanisms of how Cr(VI) orchestrates multiple mitogenic pathways to promote tumor initiation and progression remain not fully understood. Src/Ras and other growth-related pathways are shown to be key players in Cr(VI)-initiated tumor prone actions. The role of protein kinase C (PKC, an important signal transducer) in Cr(VI)-mediated carcinogenesis has not been thoroughly investigated. In this study, using human bronchial/lung epithelial cells and keratinocytes, we demonstrate that PKC activity is increased by transient or chronic Cr(VI) exposure, which plays no role in the activation of Src/Ras signaling and ROS upregulation by this metal toxin. PKC in chronic Cr(VI)-treated cells stabilizes Bcl-2 to mitigate doxorubicin (an anti-cancer drug)-mediated apoptosis. After the suppression of this kinase by GO6976 (a PKC inhibitor), the cells chronically exposed to Cr(VI) partially regain the sensitivity to doxorubicin. However, when co-suppressed PKC and Ras, the chronic Cr(VI)-treated cells become fully responsive to doxorubicin and are unable to be transformed. Taken together, our study provides a new insight into the mechanisms, in which PKC is an indispensable player and cooperates with other mitogenic pathways to achieve Cr(VI)-induced carcinogenesis as well as to establish drug resistance. The data also suggest that active PKC can serve as a potential biomarker for early detection of health damages by Cr(VI) and therapeutic target for developing new treatments for diseases caused by Cr(VI).

Interplay of the ubiquitin proteasome system and the innate immune response is essential for the replication of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNß) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNß. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.

[Atractylenolide I Can Induce Apoptosis of U266 Cells and Enhance Bortezomib Effect].

OBJECTIVE: To investigate the effect of atractylenolide I on proliferation and apoptosis of U266 cells, and anti-multiple myeloma effect of bortezomib. METHODS: Bortezomib, bortezomib combined atractylenolide I and atractylenolide I at different concentrations were added into U266 cells respectively, cellular proliferation toxicity was evaluated by CCK-8 assay, apoptosis and cell cycle were detected by using flow cytometry with Annexin V-FITC/PI staining. RT-PCR and Western blot analysis were used to detect the mRNA and protein levels of targeting gene Caspase-3,Caspase-9,BCL-2,BAX,JAK2,STAT3 and IL-6, respectively. RESULTS: The proliferation of U266 cells could inhibited by atractylenolide I, and the apoptosis of U266 cells could be promoted by atractylenolide I, also, which showed a dose-dependent manner(P<0.00; r=0.99). Moreover, the atractylenolide I could regulat the mitochondrial pathway(P<0.01). The combination of 2 drugs could strengther the inhibition of U266 cell proliferation significantly, and the expression level of IL-6,JAK2,STAT3 and BCL-2 mRNA and protein could be decreased by single drug and 2 drugs both(P<0.01). CONCLUSION: Atractylenolide I significantly inhibits the proliferation of U266 cells and promotes their apoptosis. At the same time, it acts synergistically with bortezomib, which may be related to mitochondrial pathway, and probably related to the regulating of IL-6, JAK2 and STAT3 gene expression in signal pathway of JAK2/STAT3.

Chronic low dose arsenic exposure preferentially perturbs mitotic phase of the cell cycle.

Environmental pollution is a big challenge for human survival. Arsenic compounds are well-known biohazard, the exposure of which is closely linked to onsets of various human diseases, particularly cancers. Upon chronically exposing to arsenic compounds, genomic integrity is often disrupted, leading to tumor development. However, the underlying mechanisms by which chronic, low dose arsenic exposure targets genetic stability to initiate carcinogenesis still remain not fully understood. In this study, human lung epithelial BEAS-2B cells and keratinocytes were treated with 0.5 µM of sodium arsenite for one month (designated as BEAS-2B-SA cells or keratinocytes-SA), and its effect on cell cycle responses was analyzed. After being arrested in mitotic phase of the cell cycle by nocodazole treatment, BEAS-2B-SA cells or keratinocytes-SA were delayed to enter next cytokinesis. The lagging exit of the cells from mitosis was accompanied by a sustained Plk1 phosphorylation, which led to a persistent activation of the mitotic regulators BubR1 and Cdc27. As the result, cyclin B1 (clnB1) degradation was attenuated. BEAS-2B-SA cells or keratinocytes-SA also expressed a constitutively active Akt. The cytogenetic analysis showed an increased numbers of aneuploidy in these cells. The suppression of Akt reversed the aberrant expressions of the mitotic regulators, delay of mitotic exit as well as chromosomal aberrations. Our findings suggest that a long-term exposure to low dose sodium arsenite aberrantly retains the catenation of mitosis, which facilitates establishing genetic instability and predisposes the cells to tumorigenesis.

A lethal synergy induced by phellinus linteus and camptothecin11 in colon cancer cells.

Side effects of anti-cancer drugs are always challenging for effective cancer treatments. The polysaccharides extracted from Phellinus linteus (PLGL) have been widely used in treating cancers. However, the mechanism by which PLGL antagonizes cancerous growth has not been fully investigated. The current study demonstrated that human colon cancer HCT116 and HT29 cells became highly susceptible to cell death when being co-treated with PLGL and low dose of camptothecin11 (CPT11, a topoisomerase inhibitor-based drug), the efficacy of which was comparable as that generated by the high dose of CPT11. However, the co-treatment, unlike high doses of CPT11, was not cytotoxic to the control immortalized colon Caco-2 cells. The co-treatment caused high percentages of the colon cancer cells to accumulate in S phase of the cell cycle, which was also seen in the same cells received the high dose of CPT11 treatment. Chk1 was phosphorylated, and then rapidly degraded in the cancer cells treated with the high dose of CPT11 or co-treatment, but not in the cells treated with PLGL alone or low doses of CPT11. PLGL appeared enhancing CPT11 inhibitory effect on topoisomerase, and Chk1 degradatopm in the cancer cells. Furthermore, cyclin E (clnE) became unstable at the transcription level in co-treated or PLGL-treated colon cancer cells. The data suggested that PLGL functions in two ways to achieve its lethal synergy with CPT11 in colon cancer cells. Our findings are of potential significance as PLGL represents a promising medicine for overcoming the side effects of CPT11 and perhaps also for improving other CPTs-based regimens.

Suppression of PKC causes oncogenic stress for triggering apoptosis in cancer cells.

Gain of functional mutations in ras occurs in more than 30% of human malignancies and in particular 90% of pancreatic cancer. Mutant ras, via activating multiple effector pathways, not only promote cell growth or survival, but also apoptosis, depending upon cell types or circumstances. In order to further study the mechanisms of apoptosis induced by oncogenic ras, we employed the ras loop mutant genes and demonstrated that Akt functioned downstream of Ras in human pancreatic cancer or HPNE cells ectopically expressing mutated K-ras for the induction of apoptosis after the concurrent suppression of PKC α and ß. In this apoptotic process, the redox machinery was aberrantly switched on in the pancreatic cancer cells as well as prostate cancer DU145 cells. p73 was phosphorylated and translocated to the nucleus, accompanied with UPR activation and induction of apoptosis. The in vitro results were corroborated by the in vivo data. Thus, our study indicated that PKC α and ß appeared coping with oncogenic Ras or mutated Akt to maintain the balance of the homeostasis in cancer cells. Once these PKC isoforms were suppressed, the redox state in the cancer cells was disrupted, which elicited persistent oncogenic stress and subsequent apoptotic crisis.

Chromium IV exposure, via Src/Ras signaling, promotes cell transformation.

Hexavalent chromium [Cr(VI)] is a well-known environment carcinogen. The exposure of Cr(VI) through contaminated soil, air particles, and drinking water is a strong concern for the public health worldwide. While many studies have been done, it remains unclear which intracellular molecules transduce Cr(VI)-mediated carcinogenic signaling in cells to promote cancer. In this study, we demonstrated that upon Cr(VI) treatment, the intracellular receptor src was activated, which further upregulated Ras activity, leading to the augmentation of ROS and onset of ER stress in human lung epithelial BEAS-2B or keratinocytes. These cells were formed colonies in soft agar cultures following the persistent Cr(VI) treatment. Furthermore, anti-apoptotic factor Bcl-2 was upregulated and activated in the colonies. Thus, our study suggests that Cr(VI), though activating the src and Ras signaling axis, perturbs redox state and invokes ER stress for the establishment of carcinogenic actions in the cells. In this process, Bcl-2 appears playing an important role. By uncovering these intracellular targets, our study may help developing novel strategies for better environmental protection, especially in areas contaminated or polluted by Cr(VI) as well as for effective cancer treatments.

Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

Low doses of arsenic, via perturbing p53, promotes tumorigenesis.

In drinking water and in workplace or living environments, low doses of arsenic can exist and operate as a potent carcinogen. Due to insufficient understanding and information on the pervasiveness of environmental exposures to arsenic, there is an urgent need to elucidate the underlying molecular mechanisms of arsenic regarding its carcinogenic effect on human health. In this study, we demonstrate that low doses of arsenic exposure mitigate or mask p53 function and further perturb intracellular redox state, which triggers persistent endoplasmic reticulum (ER) stress and activates UPR (unfolded protein response), leading to transformation or tumorigenesis. Thus, the results suggest that low doses of arsenic exposure, through attenuating p53-regulated tumor suppressive function, change the state of intracellular redox and create a microenvironment for tumorigenesis. Our study also provides the information for designing more effective strategies to prevent or treat human cancers initiated by arsenic exposure.