Anti-TNFR2 Antibody-Conjugated PLGA Nanoparticles for Targeted Delivery of Adriamycin in Mouse Colon Cancer.

High levels of tumor necrosis factor receptor type II (TNFR2) are preferentially expressed by immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in the tumor microenvironment, as initially reported by us. There is compelling evidence that targeting TNFR2 markedly enhances antitumor immune responses. Furthermore, a broad spectrum of human cancers also expresses TNFR2, while its expression by normal tissue is very limited. We thus hypothesized that TNFR2 may be harnessed for tumor-targeted delivery of chemotherapeutic agents. In this study, we performed a proof-of-concept study by constructing a TNFR2-targeted PEGylated poly(dl-lactic-co-glycolic acid) (PLGA-PEG) nanodrug delivery system [designated as TNFR2-PLGA-ADR (Adriamycin)]. The results of in vitro study showed that this TNFR2-targeted delivery system had the properties in cellular binding and cytotoxicity toward mouse colon cancer cells. Further, upon intravenous injection, TNFR2-PLGA-ADR could efficiently accumulate in MC38 and CT26 mouse colon tumor tissues and preferentially bind with tumor-infiltrating Tregs. Compared with ADR and ISO-PLGA-ADR, the in vivo antitumor effect of TNFR2-PLGA-ADR was markedly enhanced, which was associated with a decrease of TNFR2+ Tregs and an increase of IFNγ+CD8+ cytotoxic T lymphocytes in the tumor tissue. Therefore, our results clearly show that targeting TNFR2 is a promising strategy for designing tumor-specific chemoimmunotherapeutic agent delivery system.

Deubiquitinating enzymes: potential regulators of the tumor microenvironment and implications for immune evasion.

Ubiquitination and deubiquitination are important forms of posttranslational modification that govern protein homeostasis. Deubiquitinating enzymes (DUBs), a protein superfamily consisting of more than 100 members, deconjugate ubiquitin chains from client proteins to regulate cellular homeostasis. However, the dysregulation of DUBs is reportedly associated with several diseases, including cancer. The tumor microenvironment (TME) is a highly complex entity comprising diverse noncancerous cells (e.g., immune cells and stromal cells) and the extracellular matrix (ECM). Since TME heterogeneity is closely related to tumorigenesis and immune evasion, targeting TME components has recently been considered an attractive therapeutic strategy for restoring antitumor immunity. Emerging studies have revealed the involvement of DUBs in immune modulation within the TME, including the regulation of immune checkpoints and immunocyte infiltration and function, which renders DUBs promising for potent cancer immunotherapy. Nevertheless, the roles of DUBs in the crosstalk between tumors and their surrounding components have not been comprehensively reviewed. In this review, we discuss the involvement of DUBs in the dynamic interplay between tumors, immune cells, and stromal cells and illustrate how dysregulated DUBs facilitate immune evasion and promote tumor progression. We also summarize potential small molecules that target DUBs to alleviate immunosuppression and suppress tumorigenesis. Finally, we discuss the prospects and challenges regarding the targeting of DUBs in cancer immunotherapeutics and several urgent problems that warrant further investigation.

Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy.

Thalidomide (THD), a synthetic derivative of glutamic acid, was initially used as a sedative and antiemetic until the 1960s, when it was found to cause devastating teratogenic effects. However, subsequent studies have clearly demonstrated the anti-inflammatory, anti-angiogenic, and immunomodulatory properties of thalidomide, thus providing a rationale for its current use in the treatment of various autoimmune diseases and cancers. Our group found that thalidomide can suppress the regulatory T cells (Tregs), a minor subset of CD4+ T cells (~10%) with unique immunosuppressive activity that have been shown to accumulate in the tumor microenvironment (TME) and represent a major mechanism of tumor immune evasion. Due to the low solubility of thalidomide in its present form of administration, coupled with its lack of specificity for targeted delivery and controlled drug release, it is an urgent need to find potent delivery methods that can significantly enhance its solubility, optimize the desired site of drug action, and mitigate its toxicity. In this study, the isolated exosomes were incubated with synthetic liposomes to form hybrid exosomes (HEs) that carried THD (HE-THD) with uniform size distribution. The results demonstrated that HE-THD could significantly abrogate the expansion and proliferation of Tregs induced by TNF, and this might result from blocking TNF-TNFR2 interaction. By encapsulating THD in hybrid exosomes, our drug delivery system successfully increased the solubility of THD, laying a foundation for future in vivo experiments that validate the antitumor activity of HE-THD by reducing the Treg frequency within the tumor microenvironment.

TNFR2 deficiency impairs the growth of mouse colon cancer.

Objective: Tumor necrosis factor (TNF) receptor type II (TNFR2) is expressed by a wide spectrum of tumor cells including colon cancer, non-Hodgkin lymphoma, myeloma, renal carcinoma and ovarian cancer, and its exact role remains to be fully understood. In this study, we examined the effect of genetic ablation of TNFR2 on in vitro and in vivo growth of mouse MC38 and CT26 colon cancer cells. Methods: CRISPR/Cas9 technology was used to knockout TNFR2 on mouse MC38 and CT26 colon cancer cells. In vitro growth and colony formation of wild-type (W.T.) and TNFR2 deficiency of MC38 and CT26 cells, as well as the potential mechanism, was studied. The growth of W.T. and TNFR2 deficient MC38 and CT26 tumors in mice and intratumoral CD8 CTLs were also examined. Results: TNFR2 deficiency impaired in vitro proliferation and colony formation of cancer cells. This was associated with the inhibition of protein kinase B (AKT) phosphorylation and enhanced autophagy-induced cell death. Moreover, deficiency of TNFR2 also markedly impaired in vivo growth of MC38 or CT26 in the syngeneic C57BL/6 mice or BALB/c mice, respectively, accompanied by the decrease in soluble TNFR2 levels in the circulation and the increase in the number of tumor-infiltrating IFNγ+ CD8 cells. Conclusion: TNFR2 plays a role in the growth of mouse colon cancers. Our study provides further experimental evidence to support the development of TNFR2 antagonistic agents in the treatment of cancer.

TNFR2 antagonistic antibody induces the death of tumor infiltrating CD4+Foxp3+ regulatory T cells.

BACKGROUND: TNFR2 expression is a characteristic of highly potent immunosuppressive tumor infiltrating CD4+Foxp3+ regulatory T cells (Tregs). There is compelling evidence that TNF through TNFR2 preferentially stimulates the activation and expansion of Tregs. We and others, therefore, proposed that targeting TNFR2 may provide a novel strategy in cancer immunotherapy. Several studies have shown the effect of TNFR2 antagonistic antibodies in different tumor models. However, the exact action of the TNFR2 antibody on Tregs remained understood. METHOD: TY101, an anti-murine TNFR2 antibody, was used to examine the effect of TNFR2 blockade on Treg proliferation and viability in vitro. The role of TNFR2 on Treg viability was further validated by TNFR2 knockout mice and in the TY101 antagonistic antibody-treated mouse tumor model. RESULTS: In this study, we found that an anti-mouse TNFR2 antibody TY101 could inhibit TNF-induced proliferative expansion of Tregs, indicative of an antagonistic property. To examine the effect of TY101 antagonistic antibody on Treg viability, we treated unfractionated lymph node (L.N.) cells with Dexamethasone (Dex) which was known to induce T cell death. The result showed that TY101 antagonistic antibody treatment further promoted Treg death in the presence of Dex. This led us to find that TNFR2 expression was crucial for the survival of Tregs. In the mouse EG7 lymphoma model, treatment with TY101 antagonistic antibody potently inhibited tumor growth, resulting in complete regression of the tumor in 60% of mice. The treatment with TY101 antagonistic antibody elicited potent antitumor immune responses in this model, accompanied by enhanced death of Tregs. CONCLUSION: This study, therefore, provides clear experimental evidence that TNFR2 antagonistic antibody, TY101, can promote the death of Tregs, and this effect may be attributable to the antitumor effect of TNFR2 antagonistic antibody.

Phthalate derivative DEHP disturbs the antiproliferative effect of camptothecin in human lung cancer cells by attenuating DNA damage and activating Akt/NF-κB signaling pathway.

Plasticizers/phthalates play a facilitating role in the development of cancer and help the tumor to grow and metastasize. Camptothecin (CPT) and its derivatives are known to have anticancer properties of inhibiting cell growth, promoting cell apoptosis, and increasing autophagy. Therefore, in this study, we investigated whether the presence of di(2-ethylhexyl) phthalate (DEHP) could hinder apoptosis and autophagy caused by CPT in non-small cell lung cancer (NSCLC) cells. We found that DEHP interferes with CPT-induced apoptosis and autophagy and increases the prosurvival pathway by reducing the DNA damage marker γ-H2AX and activating the Akt and NF-κB pathways. Furthermore, we also confirmed that combining DEHP with 3-MA has additive effects in inhibiting autophagy and apoptosis in NSCLC cells. Taken together, our findings show that DEHP could affect CPT-induced anticancer treatment and provide evidence to show that DEHP induces chemoresistance in CPT-based chemotherapy.

Distepharinamide, a novel dimeric proaporphine alkaloid from Diploclisia glaucescens, inhibits the differentiation and proliferative expansion of CD4+Foxp3+ regulatory T cells.

BACKGROUND: CD4+Foxp3+ regulatory T cells (Tregs) represent the primary cellular mechanism of tumor immune evasion. Elimination of Treg activity by the pharmacological agent may enhance anti-tumor immune responses. However, Treg-eliminating agents, especially those with small molecules, are rarely reported. PURPOSE: To identify small molecule inhibitors of Treg cells from natural products. METHODS: Compounds from Diploclisia glaucescens were isolated by column chromatography, and structures were identified by spectroscopic evidence and quantum calculations. The tet-On system for Foxp3-GFP expression in Jurkat T cells was generated to screen Treg inhibitors based on Foxp3 expression. The effect of the compound on TNF-induced proliferative expansion of naturally occurring Tregs (nTregs) and TGF-ß-induced generation of Tregs (iTregs) from naive CD4+ Tcells was further examined. RESULTS: A novel dimeric proaporphine alkaloid, designated as distepharinamide (DSA) with a symmetric structure isolated from the stems of D. glaucescens, restrained the doxycycline (Doxy)-induced Foxp3-tGFP expression, decreased the half-life of Foxp3 mRNA as well as reduced the mRNA levels of chemokine receptors (CCR4, CCR8 and CCR10) in Jurkat T cells with inducible Foxp3-tGFP expression. In lymphocytes or purified Tregs from wild-type C57BL/6 mice or from C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax mice, DSA markedly inhibited TNF-induced proliferative expansion of Tregs present in the unfractionated CD4+ T cells, accompanied by the down-regulation of TNFR2, CD25 and CTLA4 expression on Tregs. Furthermore, DSA potently inhibited TGF-ß-induced differentiation of Foxp3-expressing iTregs. Importantly, the expression of Foxp3 mRNA by both nTregs and iTregs was decreased by DSA treatment. Nevertheless, DSA at the same concentrations did not inhibit the proliferation of conventional CD4+ and CD8+ T cells stimulated by anti-CD3/CD28 antibodies. CONCLUSION: DSA, a novel dimeric proaporphine alkaloid, potently inhibited the expansion of nTregs and generation of iTregs. Therefore, DSA or its analogs may merit further investigation as novel immunotherapeutic agents.

Scutellarin enhances anti-tumor immune responses by reducing TNFR2-expressing CD4+Foxp3+ regulatory T cells.

One characteristic of tumor-associated CD4+Foxp3+ regulatory T cells (Tregs) is the high expression of tumor necrosis factor receptor type II (TNFR2), a receptor that mediates the decisive effect of tumor necrosis factor (TNF) in the activation and expansion of Tregs. There is increasing evidence that inhibition of TNFR2 can enhance anti-tumor immune responses. Therefore, we screened Chinese herbal extracts for their capacity to block TNF-TNFR2 interaction. The results showed that the treatment with a Chinese herb extract could inhibit TNFR2-induced biological responses in vitro, including the proliferation of TNFR2+ Tregs. Our subsequent study led to the identification of flavonoid compound scutellarin was responsible for the activity. Our results showed that scutellarin is able to disrupt the interaction of TNF-TNFR2 and inhibited the phosphorylation of p38 MAPK, a down-stream signaling component of TNFR2. Importantly, in vivo scutellarin treatment markedly enhanced the efficacy of tumor immunotherapy with CpG oligodeoxynucleotide in mouse CT26 colon cancer model. This effect of scutellarin was associated with the reduction of the number of tumor-infiltrating TNFR2-expressing Tregs and increased tumor infiltration of interferon-γ-producing CD8+ T cells. Our result also suggests that scutellarin or its analogs may be used as an adjuvant to enhance the anti-tumor effect of immunotherapeutic agent by eliminating TNFR2+ Treg activity.

Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells.

CD4+Foxp3+ regulatory T cells (Tregs) are pivotal for the inhibition of autoimmune inflammatory responses. One way to therapeutically harness the immunosuppressive actions of Tregs is to stimulate the proliferative expansion of TNFR2-expressing CD4+Foxp3+ Tregs via transmembrane TNF (tmTNF). Here, we report that two-pore channel (TPC) inhibitors markedly enhance tmTNF expression on antigen-presenting cells. Furthermore, injection of TPC inhibitors including tetrandrine, or TPC-specific siRNAs in mice, increases the number of Tregs in a tmTNF/TNFR2-dependent manner. In a mouse colitis model, inhibition of TPCs by tetrandrine markedly attenuates colon inflammation by expansion of Tregs Mechanistically, we show that TPC inhibitors enhance tmTNF levels by disrupting surface expression of TNF-α-converting enzyme by regulating vesicle trafficking. These results suggest that the therapeutic potential of TPC inhibitors is mediated by expansion of TNFR2-expressing Tregs and elucidate the basis of clinical use in the treatment of autoimmune and other inflammatory diseases.

Preferential Expansion of CD4+Foxp3+ Regulatory T Cells (Tregs) In Vitro by Tumor Necrosis Factor.

CD4+Foxp3+ regulatory T cells (Tregs) are a distinct subset of CD4 T cells that play indispensable role in the maintenance of immune homeostasis and prevention of deleterious immune responses to self-antigens. Tumor necrosis factor (TNF) is a key cytokine in the autoimmune inflammatory responses. The effect of TNF on Treg activity was extensively studied in the past decade. We for the first time reported that TNF through TNFR2 preferentially activates and expands Tregs. Our discovery is increasingly supported by the research community; however, some controversial results were reported. The differential results are likely caused by different experimental condition. A standard experiment protocol can help researchers to obtain more consistent results. In this chapter, we detail methods used to examine in vitro effect of exogenous TNF on the proliferative expansion of Tregs in unfractionated mouse CD4+ T cells. The related technic issues are analyzed and discussed.

Synergistic effect of all-trans-retinal and triptolide encapsulated in an inflammation-targeted nanoparticle on collagen-induced arthritis in mice.

Targeted delivery of nano-encapsulated anti-inflammatory agent represents a promising while challenging strategy in the treatment of rheumatoid arthritis (RA). Pro-inflammatory macrophages play a major role in the pathogenesis of RA. In this study, we investigated the effect of a macrophage-targeted pH-sensitive nanoparticle on collagen-induced arthritis (CIA) in mice. To target macrophage, all-trans-retinal was conjugated into dextran backbone through pH-sensitive hydrazone bond, then grafted with galactose (GDR). This nanoparticle was used for the encapsulation of triptolide (TPT), a potent anti-inflammatory compound isolated from Chinese herb. As expected, GDR nanoparticles preferentially accumulated in the inflammatory tissues. Treatment with GDR-TPT nanoparticles resulted in a marked decrease in the infiltration of CD3+ T cells and F4/80+ macrophages and reduction of the expression of TNF-α, IL-6 and IL-1ß in the inflamed lesions of CIA mice. Furthermore, Th1 and Th17 responses were also inhibited. Importantly, anti-arthritic effect of TPT was markedly enhanced while its toxic effect was attenuated by encapsulating with GDR. GDR by itself also had moderate effect in the inhibition of arthritis, due to its intrinsic anti-inflammatory property. Therefore, our results clearly show that GDR-TPT nanoparticle may represent a promising drug delivery system for the treatment of RA.

Unfolded Protein Response (UPR) in Survival, Dormancy, Immunosuppression, Metastasis, and Treatments of Cancer Cells.

The endoplasmic reticulum (ER) has diverse functions, and especially misfolded protein modification is in the focus of this review paper. With a highly regulatory mechanism, called unfolded protein response (UPR), it protects cells from the accumulation of misfolded proteins. Nevertheless, not only does UPR modify improper proteins, but it also degrades proteins that are unable to recover. Three pathways of UPR, namely PERK, IRE-1, and ATF6, have a significant role in regulating stress-induced physiological responses in cells. The dysregulated UPR may be involved in diseases, such as atherosclerosis, heart diseases, amyotrophic lateral sclerosis (ALS), and cancer. Here, we discuss the relation between UPR and cancer, considering several aspects including survival, dormancy, immunosuppression, angiogenesis, and metastasis of cancer cells. Although several moderate adversities can subject cancer cells to a hostile environment, UPR can ensure their survival. Excessive unfavorable conditions, such as overloading with misfolded proteins and nutrient deprivation, tend to trigger cancer cell death signaling. Regarding dormancy and immunosuppression, cancer cells can survive chemotherapies and acquire drug resistance through dormancy and immunosuppression. Cancer cells can also regulate the downstream of UPR to modulate angiogenesis and promote metastasis. In the end, regulating UPR through different molecular mechanisms may provide promising anticancer treatment options by suppressing cancer proliferation and progression.

Half-sandwich Ru(η6-p-cymene) complexes featuring pyrazole appended ligands: Synthesis, DNA binding and in vitro cytotoxicity.

Organometallic Ru(II)-arene complexes have emerged as potential alternatives to platinum appended agents due to their wide range of interesting features such as stability in solution and solid, significant activity, less toxicity and hydrophobic property of arene moiety, etc. Hence, a series of Ru(II)-p-cymene complexes, [(η6-p-cymene)Ru(η2-N,N-L1)Cl]Cl (1), [(η6-p-cymene)Ru(η1-N-L2)Cl2] (2) and [(η6-p-cymene)Ru(η1-N-L3)Cl2] (3) were prepared from pyrazole based ligands [2-(1H-pyrazol-3-yl)pyridine (L1), 3-(furan-2-yl)-1H-pyrazole (L2) and 3-(thiophen-2-yl)-1H-pyrazole (L3)], and [RuCl2-(η6-p-cymene)] dimer. The new Ru(II)-p-cymene complexes were well characterized by elemental analysis, and spectroscopic (FT-IR, UV-Visible, 1H NMR, 13C NMR and mass) and crystallographic methods. The Ru(II)-p-cymene complexes (1-3) were found to adopt their characteristic piano stool geometry around Ru(II) ion. The calf thymus DNA (CT-DNA) binding ability of the new complexes was investigated by electronic absorption spectroscopic titration and viscosity methods. The molecular docking study results showed that complex 1 strongly bound with targeted biomolecules than 2 and 3. Docked poses of bidentate pyrazole based Ru(II)-p-cymene complex 1 revealed that the complex formed a crucial guanine N7 position hydrogen bond with DNA receptor. Complexes 1-3 might hydrolyze under physiological conditions and form aqua complexes 4-8, and docking calculations showed that the aqua complexes bound strongly with the receptors than original complexes. The in vitro cytotoxicity of the Ru(II)-p-cymene complexes and cisplatin was evaluated against triple negative breast cancer (TNBC) MDA-MB-231 cells. Our results showed that the inhibitory effect of bidentate pyrazole based Ru(II)-p-cymene complex 1 on the growth of breast cancer cells was superior to other tested complexes.

Reduced camptothecin sensitivity of estrogen receptor-positive human breast cancer cells following exposure to di(2-ethylhexyl)phthalate (DEHP) is associated with DNA methylation changes.

Di(2-ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα-positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF-7 (ERα-dependent) and MDA-MB-231 (ERα-independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT-induced formation of reactive oxygen species in ERα-positive MCF-7 cells, but not in ERα-negative MDA-MB-231 cells. DEHP also significantly protected MCF-7 cells against the genotoxicity of CPT. Genome-wide DNA methylation profiling revealed that after 48 hours of exposure to 100 µM DEHP, MCF-7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP-exposed MCF-7 cells is probably mediated by epigenetic changes, especially through Wnt/ß-catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT-induced anti-growth of MCF-7 cells. In summary, DEHP exposure induces acquired CPT-resistance in breast cancer cells and epigenetic changes associated with Wnt/ß-catenin signaling activation are probably depending on an ER-positive status.

Phytochemical naphtho[1,2-b] furan-4,5­dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer.

BACKGROUND: Phytochemical naphtho[1,2-b] furan-4,5­dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PURPOSE: To examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. STUDY DESIGN: The anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. RESULTS: NFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. CONCLUSION: These findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress.

Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Moderate activation of UPR is required for maintaining endoplasmic reticulum (ER) homeostasis and profoundly contributes to tumorigenesis. Defects in UPR signaling are implicated in the attenuation of various malignant phenotypes including cell proliferation, migration, and invasion, as well as angiogenesis. This suggests UPR as a promising target in cancer therapy. The pharmacological effects of the plant Scindapsus cf. hederaceus on human cancer cell lines is not understood. In this study, we identified an ethyl acetate extract from Scindapsus cf. hederaceus (SH-EAE), which markedly altered the protein expression of UPR-related genes in human non-small cell lung cancer (NSCLC) cells. Treatment with the SH-EAE led to the dose-dependent suppression of colony forming ability of both H1299 and H460 cells, but not markedly in normal bronchial epithelial BEAS-2B cells. SH-EAE treatment also attenuated the migration and invasion ability of H1299 and H460 cells. Moreover, SH-EAE strikingly suppressed the protein expression of two ER stress sensors, including inositol requiring enzyme-1α (IRE-1α) and protein kinase R-like ER kinase (PERK), and antagonized the induction of C/EBP homologous protein (CHOP) expression by thapsigargin, an ER stress inducer. SH-EAE induced the formation of massive vacuoles which are probably derived from ER. Importantly, SH-EAE impaired the formation of intersegmental vessels (ISV) in zebrafish larvae, an index of angiogenesis, but had no apparent effect on the rate of larval development. Together, our findings demonstrate, for the first time, that the ability of SH-EAE specifically targets the two sensors of UPR, with significant anti-proliferation and anti-migration activities as a crude extract in human NSCLC cells. Our finding also indicates potential applications of SH-EAE in preventing UPR activation in response to Tg-induced ER stress. We suggest that SH-EAE attenuates UPR adaptive pathways for rendering the NSCLC cells intolerant to ER stress.

The Impact of Di(2-ethylhexyl)phthalate on Cancer Progression.

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer, mainly serves as an additive to render polyvinyl chloride (PVC) soft and flexible. PVC plastics have become ubiquitous in our modern society. Yet, the leaching of DEHP from PVC-based consumables ultimately results in the deposition in certain tissues via inadvertent applications. Health risks for human populations exposed to DEHP has been assumed by studies on rodents and other species, including the DEHP-induced developmental dysregulation, reproductive impairments, tumorigenesis, and diseases in a transgenerational manner. In this review, we comprehensively summarize the accumulated literature regarding the multifaceted roles of DEHP in the activation of the nuclear receptors, the alteration of the redox homeostasis, epigenetic modifications and the acquisition of chemoresistance.

The Potential Role of Krüppel-Like Zinc-Finger Protein Glis3 in Genetic Diseases and Cancers.

Gli-similar 3 (Glis3) belongs to a Glis subfamily of Krüppel-like zinc-finger transcription factors characterized to regulate a set of downstream targets essential for cellular functions, including pancreatic development, ß-cell maturation and maintenance, and insulin production. Examination of the DNA-binding domain of Glis3 reveals that this domain contains a repeated cysteine 2/histidine 2 (Cys2/His2) zinc-finger motif in the central region where the recognized DNA sequence binds. The loss of the production of pancreatic hormones, such as insulin 1 and 2, is linked to the down-regulation of ß cells-related genes and promotes the apoptotic death of ß cells found in mutant Glis3. Although accumulating studies converge on the Glis3 functioning in ß cells, recently, there have been developments in the field of Glis3 using knockdown/mutant mice to better understand the role of Glis3 in diseases. The Glis3 mutant mice have been characterized for their propensity to develop congenital hypothyroidism, polycystic kidney disease, and some types of cancer. In this review, we attempt to comprehensively summarize the knowledge of Glis3, including its structure and general function in cells. We also collected and organized the academic achievements related to the possible mechanisms of Glis3-related diseases.

A 4-Phenoxyphenol Derivative Exerts Inhibitory Effects on Human Hepatocellular Carcinoma Cells through Regulating Autophagy and Apoptosis Accompanied by Downregulating α-Tubulin Expression.

Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B)-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX), an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the phosphorylation of protein kinase B (Akt) and the level of survivin on Ha22T cells, which may confer a resistance toward 4-HPPP. Notably, the blockade of extracellular signal-regulated kinases (ERK), but not Akt, enhanced the cytotoxicity of 4-HPPP against Ha22T cells, indicating the pro-survival role of ERK in 4-HPPP-induced anti-HCC effect. Our present work suggests that selective anti-HCC activity of 4-HPPP acts through induction of DNA damage. Accordingly, the combination of ERK inhibitor may significantly enhance the anti-cancer effect of 4-HPPP for those HCC cells which overexpress ERK in the future.

Correction: Chon-Kit Chou, et al. The Regulations of Deubiquitinase USP15 and Its Pathophysiological Mechanisms in Diseases. Int. J. Mol. Sci. 2017, 18, 483.

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