Luguo Sun

CaMK IV phosphorylates prohibitin 2 and regulates prohibitin 2-mediated repression of MEF2 transcription.

n Abstractn n Prohibitin 2 (PHB2) is an evolutionarily conserved and ubiquitously expressed multifunctional protein which is present in various cellular compartments including the nucleus. However, mechanisms underlying various functions of PHB2 are not fully explored yet. Previously we showed that PHB2 interacts with Akt and inhibits muscle differentiation by repressing the transcriptional activity of both MyoD and MEF2. Here we show that Calcium/Calmodulin-dependent kinase IV (CaMK IV) specifically binds to the C terminus of PHB2 and phosphorylates PHB2 at serine 91. Ectopic expression of CaMK IV and PHB2 in C2C12 cells results effectively in decreased PHB2-mediated repression of MEF2-dependent gene expression. Conversely, PHB2 mutant (S91A) resistant to CaMK IV phosphorylation has less effective in relieving the inhibition of MEF2 transcription by PHB2. Our findings suggest that CaMK IV interacts with and regulates PHB2 through phosphorylation, which could be one of the mechanisms underlying the CaMK-mediated activation of MEF2.n n

Cardamonin inhibited cell viability and tumorigenesis partially through blockade of testes-specific protease 50-mediated nuclear factor-kappaB signaling pathway activation.

Previous studies have shown that testes-specific protease 50 (TSP50), a pro-oncogene overexpressed in many types of tumors, could promote cell proliferation, invasion, tumorigenesis, and tumor metastasis, suggesting that it is a potential cancer therapeutic target in drug discovery. Here, a luciferase assay system driven by the TSP50 gene promoter was used to screen the inhibitor of expression of TSP50. The study found that cardamonin, a flavone compound, could efficiently inhibit the expression of TSP50 in both mRNA and protein levels. Further results revealed that cardamonin also efficiently inhibited the viability of TSP50 high-expressing cancer cells by inducing G2/M-phase arrest and mitochondrial-dependent apoptosis. Surprisingly, knocking down the expression of TSP50 gene had the same effects as treatment with cardamonin. Moreover, it has been found that cardamonin had an inhibitory potency on TSP50 high-expressing tumor growth in vivo. In contrast, overexpression of TSP50 greatly decreased the cell sensitivity to the inhibitory effect of cardamonin and reversed the decreased tumor-inhibitory effect of cardamonin. Additionally, both TSP50 interference and treatment with cardamonin could suppress p65 nuclear translocation, and overexpression of TSP50 reversed the suppressive effect of cardamonin on p65 nuclear translocation. Taken together, these results suggest that cardamonin inhibited cell viability and tumorigenesis at least partially via blocking the activation of TSP50-mediated nuclear factor-kappaB signaling pathway, and cardamonin may be a promising anticancer drug candidate in the development of a novel agent for TSP50 high-expressing cancer cells.

Near-infrared conjugated polymers for photoacoustic imaging-guided photothermal/chemo combination therapy

Conjugated polymers (CPs) with intensive near-infrared (NIR) absorption and high photothermal conversion efficiency (PCE) have emerged as a new generation of photothermal therapy (PTT) and photoacoustic imaging (PAI) agents for cancer therapy. PTT + chemotherapy has been identified as a powerful modality to offer synergistic effects in the destruction and monitoring of cancer tissues. In this study, diketopyrrolopyrrole-based polymers (DPP) were designed through a combination of donor-acceptor moieties. Then, doxorubicin (DOX) and DPP were co-encapsulated in tocopheryl polyethylene-glycol-succinate-cholesterol (TPGS-CHO) copolymers to build a combined theranostic system for tumor treatment. These combined NPs with high PCE (∼50%) and strong (NIR) absorption exhibit excellent real-time photoacoustic imaging detection and synergistic cancer inhibition.

Efficient Cancer Regression by a Thermosensitive Liposome for Photoacoustic Imaging-Guided Photothermal/Chemo Combinatorial Therapy

The capacity to specifically destroy cancer cells while avoiding normal tissue is urgently desirable in cancer treatment. Herein, a photothermal-trigger-released system serves as a photoacoustic imaging agent constructed by entrapping diketopyrrolopyrrole-based conjugated polymers and curcumin in a poly(ethylene glycol) (PEG)-protected thermoresponsive liposomal phospholipid bilayer. This lipid nanostructure can improve the bioavailability of hydrophobic agents for photothermal treatment with high efficiency and deliver the anticancer drug curcumin to the tumor site actuated by near-infrared (NIR) irradiation. A significantly enhanced combined therapeutic effect to HepG2 tumor-bearing mice was acquired in contrast to the result of single therapy alone. These liposomes with the capability of photoacoustic imaging, greater EPR-induced accumulation in tumor sites, and hyperthermia ablation for photothermal chemotherapy show potential for photoacoustic imaging-guided photothermal/chemo combined therapeutic applications.

Periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in the TPA- and IMQ-induced psoriasis-like mouse models

Psoriasis is a multifactorial skin disease that inconveniences many patients. Considering the side effects and drug resistance of the current therapy, it is urgent to discover more effective and safer anti-psoriatic drugs. In the present study, we screened over 250 traditional Chinese medicine compounds for their ability to inhibit the cell viability of cultured human HaCaT keratinocytes, a psoriasis-relevant in vitro model, and found that periplogenin was highly effective. Mechanistic studies revealed that apoptosis and autophagy were not induced by periplogenin in HaCaT cells. However, periplogenin caused PI to permeate into cells, increased lactate LDH release and rapidly increased the number of necrotic cells. Additionally, the typical characteristics of necrosis were observed in the periplogenin-treated HaCaT cells. Notably, the necroptosis inhibitor Nec-1 and NSA were able to rescue the cells from necrotic cell death, supporting that necroptosis was involved in periplogenin-induced cell death. Furthermore, the ROS levels were elevated in the periplogenin-treated cells, NAC (an antioxidant) and Nec-1 could inhibit the ROS levels, and NAC could attenuate necroptotic cell death, indicating that the periplogenin-induced necroptotic cell death was mediated by oxidative stress. More importantly, in the murine models of TPA-induced epidermal hyperplasia and IMQ-induced skin inflammation, topical administration of periplogenin ameliorated skin lesions and inflammation. In sum, our results indicate, for the first time, that periplogenin is a naturally occurring compound with potent anti-psoriatic effects in vitro and in vivo, making it a promising candidate for future drug research.

Virtual screening and experimental validation of novel histone deacetylase inhibitors

BackgroundHistone deacetylases (HDACs) are promising therapeutic targets for the treatment of cancer, diabetes and other human diseases. HDAC inhibitors, as a new class of potential therapeutic agents, have attracted a great deal of interest for both research and clinical applications. Increasing efforts have been focused on the discovery of HDAC inhibitors and some HDAC inhibitors have been approved for use in cancer therapy. However, most HDAC inhibitors, including the clinically approved agents, do not selectively inhibit the deacetylase activity of class I and II HDAC isforms, and many suffer from metabolic instability. This study aims to identify new HDAC inhibitors by using a high-throughput virtual screening approach.MethodsAn integration of in silico virtual screening and in vitro experimental validation was used to identify novel HDAC inhibitors from a chemical database.ResultsA virtual screening workflow for HDAC inhibitors were created by integrating ligand- and receptor- based virtual screening methods. Using the virtual screening workflow, 22 hit compounds were selected and further tested via in vitro assays. Enzyme inhibition assays showed that three of the 22 compounds had HDAC inhibitory properties. Among these three compounds, ZINC12555961 significantly inhibited HDAC activity. Further in vitro experiments indicated that ZINC12555961 can selectively inhibit proliferation and promote apoptosis of cancer cells.ConclusionsIn summary, our study presents three new and potent HDAC inhibitors and one of these HDAC inhibitors shows anti-proliferative and apoptosis-inducing activity against various cancer cell lines. These results suggest that the developed virtual screening workflow can provide a useful source of information for the screening and validation of new HDAC inhibitors. The new-found HDAC inhibitors are worthy to further and more comprehensive investigations.

25-methoxyl-dammarane-3β, 12β, 20-triol and artemisinin synergistically inhibit MDA-MB-231 cell proliferation through downregulation of testes-specific protease 50 (TSP50) expression.

While the incidence of cancer continues to increase, the current therapeutic options remain imperfect. Therefore, there is an urgent need to discover new targeted anti-cancer therapies. Testes-specific protease 50 (TSP50) is abnormally expressed in most cancer tissues and downregulation of TSP50 expression can reduce cell proliferation and induce cell apoptosis, which makes it a potential target for cancer therapy. In this study, we constructed a firefly luciferase reporter pGL3-TSP50-3′-UTR as a drug screening model to screen potential candidate compounds that target TSP50 mRNA. We identified the compound 7P3A, which consists of 70xa0% 25-methoxyl-dammarane-3β, 12β, 20-triol and 30xa0% artemisinin, as being capable of inhibiting the TSP50-3′-UTR reporter activity, as well as the expression of TSP50. Further investigation revealed that 7P3A could inhibit MDA-MB-231 cell proliferation and induce cell cycle arrest, and over-expression of TSP50 partially reversed the effect of 7P3A. In vivo investigation showed that 7P3A could inhibit tumor growth in a xenograft model of breast cancer. These results suggest that 7P3A exhibits anti-cancer effects, in part, through downregulation of TSP50 expression.

20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol negatively regulates activation of STAT3 and ERK pathways and exhibits anti-cancer effects in HepG2 cells

The pro-inflammatory cytokine interleukin 6 (IL-6), via activating its downstream JAK/STAT3 and Ras/ERK signaling pathways, is involved in cell growth, proliferation and anti-apoptotic activities in various malignancies. To screen inhibitors of IL-6 signaling, we constructed a STAT3 and ERK dual-pathway responsive luciferase reporter vector (Co.RE). Among several candidates, the natural compound 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol (25-OCH3-PPD, GS25) was identified to clearly inhibit the luciferase activity of Co.RE. GS25 was confirmed to indeed inhibit activation of both STAT3 and ERK pathways and expression of downstream target genes of IL-6, and to predominantly decrease the viability of HepG2 cells via induction of cell cycle arrest and apoptosis. Interestingly, GS25 showed preferential inhibition of HepG2 cell viability relative to normal liver L02 cells. Further investigation showed that GS25 could not induce apoptosis and block activation of STAT3 and ERK pathways in L02 cells as efficiently as in HepG2 cells, which may result in differential effects of GS25 on malignant and normal liver cells. In addition, GS25 was found to potently suppress the expression of endogenous STAT3 at a higher concentration and dramatically induce p38 phosphorylation in HepG2 cells, which could mediate its anti-cancer effects. Finally, we demonstrated that GS25 also inhibited tumor growth in HepG2 xenograft mice. Taken together, these findings indicate that GS25 elicits its anti-cancer effects on HepG2 cells through multiple mechanisms and has the potential to be used as an inhibitor of IL-6 signaling. Thus, GS25 may be developed as a treatment for hepatocarcinoma with low toxicity on normal liver tissues as well as other inflammation-associated diseases.

Anti-biofilm Activities from Bergenia crassifolia Leaves against Streptococcus mutans

Streptococcus mutans has been reported as a primary cariogenic pathogen associated with dental caries. The bacteria can produce glucosyltransferases (Gtfs) to synthesize extracellular polysaccharides (EPSs) that are known as virulence factors for adherence and formation of biofilms. Therefore, an ideal inhibitor for dental caries is one that can inhibit planktonic bacteria growth and prevent biofilm formation. Bergenia crassifolia (L.), widely used as a folk medicine and tea beverage, has been reported to have a variety of bioactivities. The present study aimed to explore the effect of B. crassifolia (L.) leaf extracts on the biofilm of Streptococcus mutans. The B. crassifolia (L.) leaf extracts showed inhibitory effects by decreasing viability of bacteria within the biofilm, as evidenced by the XTT assay, live/dead staining assay and LDH activity assay, and could decrease the adherence property of S. mutans through inhibiting Gtfs to synthesize EPSs. In addition, the reduced quantity of EPSs and the inhibition of Gtfs were positively correlated with concentrations of test samples. Finally, the MTT assay showed that the extracts had no cytotoxicity against normal oral cells. In conclusion, the extracts and sub-extracts of B. crassifolia leaves were found to be antimicrobial and could reduce EPS synthesis by inhibiting activities of Gtfs to prevent bacterial adhesion and biofilm formation. Therefore, B. crassifolia leaves have potential to be developed as a drug to prevent and cure dental caries.

Prohibitin 2 localizes in nucleolus to regulate ribosomal RNA transcription and facilitate cell proliferation in RD cells

Prohibitin 2 (PHB2), as a conserved multifunctional protein, is traditionally localized in the mitochondrial inner membrane and essential for maintenance of mitochondrial function. Here, we investigated the role of PHB2 in human rhabdomyosarcoma (RMS) RD cells and found substantial localization of PHB2 in the nucleolus. We demonstrated that PHB2 knockdown inhibited RD cell proliferation through inducing cell cycle arrest and suppressing DNA synthesis. Meanwhile, down-regulation of PHB2 also induced apoptosis and promoted differentiation in fractions of RD cells. In addition, PHB2 silencing led to altered nucleolar morphology, as observed by transmission electron microscopy, and impaired nucleolar function, as evidenced by down-regulation of 45S and 18S ribosomal RNA synthesis. Consistently, upon PHB2 knockdown, occupancy of c-Myc at the ribosomal DNA (rDNA) promoter was attenuated, while more myoblast determination protein 1 (MyoD) molecules bound to the rDNA promoter. In conclusion, our findings suggest that nucleolar PHB2 is involved in maintaining nucleolar morphology and function in RD cells by regulating a variety of transcription factors, which is likely to be one of the underlying mechanisms by which PHB2 promotes tumor proliferation and represses differentiation. Our study provides new insight into the pathogenesis of RMS and novel characterizations of the highly conserved PHB2 protein.