A non-genetic engineering platform for rapidly generating and expanding cancer-specific armed T cells.

BACKGROUND: Cancer-specific adoptive T cell therapy has achieved successful milestones in multiple clinical treatments. However, the commercial production of cancer-specific T cells is often hampered by laborious cell culture procedures, the concern of retrovirus-based gene transfection, or insufficient T cell purity. METHODS: In this study, we developed a non-genetic engineering technology for rapidly manufacturing a large amount of cancer-specific T cells by utilizing a unique anti-cancer/anti-CD3 bispecific antibody (BsAb) to directly culture human peripheral blood mononuclear cells (PBMCs). The anti-CD3 moiety of the BsAb bound to the T cell surface and stimulated the differentiation and proliferation of T cells in PBMCs. The anti-cancer moiety of the BsAb provided these BsAb-armed T cells with the cancer-targeting ability, which transformed the naïve T cells into cancer-specific BsAb-armed T cells. RESULTS: With this technology, a large amount of cancer-specific BsAb-armed T cells can be rapidly generated with a purity of over 90% in 7 days. These BsAb-armed T cells efficiently accumulated at the tumor site both in vitro and in vivo. Cytotoxins (perforin and granzyme) and cytokines (TNF-α and IFN-γ) were dramatically released from the BsAb-armed T cells after engaging cancer cells, resulting in a remarkable anti-cancer efficacy. Notably, the BsAb-armed T cells did not cause obvious cytokine release syndrome or tissue toxicity in SCID mice bearing human tumors. CONCLUSIONS: Collectively, the BsAb-armed T cell technology represents a simple, time-saving, and highly safe method to generate highly pure cancer-specific effector T cells, thereby providing an affordable T cell immunotherapy to patients.

Total Syntheses, Absolute Configurations, and Cytotoxicity Evaluation of Ugonstilbenes A, B, and C from the Rhizomes of Helminthostachys zeylanica.

This study describes the first and efficient syntheses of the naturally occurring ugonstilbenes A, B, and C. The stilbene skeleton was prepared using the Horner-Wadsworth-Emmons reaction. On the basis of their specific rotations, the absolute configurations of ugonstilbenes A and C were both determined to be R, while the absolute configuration of ugonstilbene B was determined as 4aS,9aR. The synthesized compounds showed cytotoxic activities against selected human cancer cell lines.

Arenarosides A-G, Polyhydroxylated Oleanane-Type Saponins from Polycarpaea arenaria and their Cytotoxic and Antiangiogenic Activities.

Seven new polyhydroxylated oleanane-type triterpene saponins, arenarosides A-G (1-7), together with four known compounds, were isolated from an ethanol extract of the aerial parts of the Vietnamese plant Polycarpaea arenaria. The chemical structures of the newly isolated oleanane saponins were elucidated on the basis of spectroscopic and spectrometric analysis, especially 2D NMR and HRMS. Biological evaluation revealed that 3, 4, 6, and 7 showed moderate activities against four human cancer cell lines (A549, HTC116, PC3, and RT112) with IC50 values of 6.0-9.9 µM, and 3, 4, 5, and 7 also displayed promising antiangiogenesis effects with IC50 values <5 µM in the test system used. Among the isolates, arenaroside D (4) exhibited the most potent inhibitory effects, not only in cancer cell proliferation but also in angiogenic activities. Preliminary SAR studies revealed that the presence of an acetyl group at C-22 in oleanane-type triterpene saponins increases these bioactivities.

Simply Mixing Poly Protein G with Detection Antibodies Enhances the Detection Limit and Sensitivity of Immunoassays.

An insufficient amount of detection antibodies bound to their antigens usually limits the sensitivity of immunoassays. Here, we describe a simple method to improve the detection limit and sensitivity of various immunoassays by mixing detection antibodies with a soluble poly protein G (named 8pG). 8pG was developed by fusing eight repeated fragment crystallizable (Fc) binding domains of streptococcal protein G to a linear polymer. Simply mixing detection antibodies with 8pG to form an antibody/8pG complex largely increased the accumulation of detection antibody to target molecules, which dramatically enhanced the sensitivity in direct ELISA, sandwich ELISA, Western blot, and flow cytometry systems, separately. The detection limit of Western blot for low-abundance PEGylated interferon (Pegasys) and recombinant human CTLA4 (rhCTLA4) improved by at least 13-fold and 31-fold, respectively, upon mixing detection antibodies with 8pG. Moreover, the nanoscale size of the antibody/8pG complex did not influence the granularity and dimension of target cells in the flow cytometry system. Collectively, we provide a quick and easy-to-operate method to make various immunoassays to sensitively detect low-abundance target molecules by just mixing their detection antibodies with 8pG.

Interleukin-1 Receptor Type 2 Acts with c-Fos to Enhance the Expression of Interleukin-6 and Vascular Endothelial Growth Factor A in Colon Cancer Cells and Induce Angiogenesis.

Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor of exogenous IL-1; however, its intracellular activity is poorly understood. We previously demonstrated that IL1R2 intracellularly activates the expression of several proinflammatory cytokines and affects cell migration. In this study, we found that intracellular IL1R2 expression was increased in human colorectal cancer cells (CRCs) compared with normal colon cells. We also observed that the mRNA levels of IL1R2 were highly correlated with IL-6 in tumor tissues of CRC patients. By modulating its expression in CRC cells, we verified that enhanced IL1R2 expression transcriptionally activated the expression of IL-6 and VEGF-A. Conditioned medium harvested from IL1R2-overexpressing CRC cells contained higher levels of IL-6 and VEGF-A than that from vector control cells and significantly enhanced the proliferation, migration, and tube formation of cultured endothelial cells. We further demonstrated a positive association of intracellular IL1R2 levels with tumor growth and microvessel density in xenograft mouse models. These results revealed that IL1R2 activates the expression of angiogenic factors. Mechanistically, we revealed that IL1R2 complexes with c-Fos and binds to the AP-1 site at the IL-6 and VEGF-A promoters. Together, these results reveal a novel function of intracellular IL1R2 that acts with c-Fos to enhance the transcription of IL-6 and VEGF-A, which promotes angiogenesis in CRC.

Three New Sesquiterpene Aryl Esters from the Mycelium of Armillaria mellea.

Three new sesquiterpene aryl esters and eight known compounds were isolated from the EtOH extract of the mycelium of Armillaria mellea. The structures of new compounds were established by analysis of their spectroscopic data. Some of the isolates showed cytotoxicity to a variety of cancer cell lines, including MCF-7, H460, HT-29, and CEM.

Rational design of synthetically tractable HDAC6/HSP90 dual inhibitors to destroy immune-suppressive tumor microenvironment.

INTRODUCTION: The tumor microenvironment is mainly flooded with immunosuppressive cells and inhibitory cytokines, resulting in the inability of effective immune cells to infiltrate and recognize tumors and even the loss of anti-cancer ability. OBJECTIVES: We propose a novel HDAC6/HSP90 dual inhibitory strategy as well as a chemoimmunotherapeutic agent that does not only kill tumor cells but also destroys the tumor microenvironment and enhances anti-cancer immunity. METHODS: A hybrid scaffold construction approach was leveraged to furnish a series of rationally designed resorcinol-based hydroxamates as dual selective HDAC6/HSP90 inhibitors. The drug design campaign commenced with a fragment recruitment process to pinpoint validated structural units to inhibit HDAC6 and HSP90, followed by their installation in flexible HDAC inhibitory templates via an efficient and facile multistep synthetic route. Subsequent evaluations identified a strikingly potent selective HDAC6/HSP90 dual inhibitor (compound 17) via molecular and biological analysis in vitro and in vivo. RESULTS: Compound 17 exhibited not only direct cytotoxicity to cancer cells but also downregulated immune checkpoints (PD-L1 and IDO) expression in tumors via the inhibition of STAT1 pathway and degradation of oncogene proteins (Src, AKT, Rb, and FAK), leading to in vivo tumor growth inhibition. These multiple effects enabled the effector T cells to largely infiltrate into the tumor region and release granzyme B to kill cancer cells. In addition, compound 17 also decreased TGF-ß secretion from normal cells, resulting in the systemic reduction of immunosuppressive regulatory T cells. Delightfully, a cocktail treatment of compound 17 and anti-PD-1 antibodies demonstrated synergistic efficacy to eliminate solid tumors with 83.9% of tumor growth inhibition. CONCLUSION: In summary, the impressive activity profile of compound 17, as an effective anticancer agent and a potential immunosensitizer, forecasts the application of HDAC6/HSP90 dual inhibitory strategy to overcome the immunosuppressive tumor microenvironment.

Comparing the Therapeutic Mechanism and Immune Response of Human and Mouse Mesenchymal Stem Cells in Immunocompetent Mice With Acute Liver Failure.

Current mesenchymal stem cell (MSC) research is based on xenotransplantation of human MSCs (hMSCs) in immunodeficient mice and cannot comprehensively predict MSC repair mechanisms and immunomodulatory effects in damaged tissue. This study compared the therapeutic efficacy, mechanisms, and immune response of hMSCs and mouse MSCs (mMSCs) in immunocompetent mice with CCl4-induced acute liver failure. mMSCs maintained F4/80+ hepatic macrophage recruitment into the damaged liver region, increased IL-6-dependent hepatocyte proliferation, and reduced inflammatory TNF-α cytokine secretion. Moreover, mMSCs reduced α-SMA+ myofibroblast activation by lowering TGF-ß1 accumulation in damaged liver tissue. In contrast, hMSCs lowered TNF-α and TGF-ß1 by reducing the recruitment of F4/80+ hepatic macrophages, which lost the ability to remove debris and induce IL-6 liver regeneration. Finally, hMSCs, but not mMSCs, caused a significant antibody response in immunocompetent mice; therefore, hMSCs are unsuitable for long-term MSC studies. This comparative study provides reference information for further MSC studies of immunocompetent mice.

Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1.

The synthesis, cytotoxicity, inhibition of tubulin polymerization data and anti-angiogenetic effects of seven 1,5-disubstituted 1,2,3-triazole analogs and two 1,4-disubstituted 1,2,3-triazole analogs of combretastatin A-1 (1) are reported herein. The biological studies revealed that the 1,5-disubstituted 1,2,3-triazoles 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diol (6), 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diamine (8) and 5-(2,3-difluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (9) were the three most active compounds regarding inhibition of both tubulin polymerization and angiogenesis. Molecular modeling studies revealed that combretastatins 1 and 2 and analogs 5-11 could be successfully docked into the colchicine binding site of α,ß-tubulin.

Cytotoxic apigenin derivatives from Chrysopogon aciculatis.

Four new apigenin derivatives, 7-de-O-methylaciculatin, 8-C-ß-D-boivinopyranosylapigenin, aciculatinone, and 4'-O-glucosylaciculatin, along with eight known compounds, apigenin-8-carbaldehyde, kaempferol, tricin, taxifolin, 6,7,4'-trihydroxyflavone, trans-oxyresveratrol, aciculatin, and luteolin-7-sulfate, were isolated from an ethanolic extract of Chrysopogon aciculatis. Their chemical structures were elucidated by spectroscopic methods. Among the known compounds, the natural occurrence of apigenin-8-carbaldehyde and luteolin-7-sulfate is demonstrated for the first time. Some of the isolates were evaluated for cytotoxic activity against human cancer cell lines including MCF-7, H460, HT-29, and CEM.

New alkaloids and cytotoxic principles from Sinomenium acutum.

Two new alkaloids, 2-demethyl-oxypalmatine (1) and 5-ethoxycarbonylsinoracutine (2), were isolated from the rhizomes of Sinomenium acutum, along with thirty-four known compounds. Cytotoxicity of the isolated compounds was examined for the MCF-7, H460, HT-29, and CEM human cancer cell lines. Dauriporphine (16), 6-O-demethylmenisporphine (17), bianfugecine (18), menisporphine (19), and 6-O-demethyldauriporphine (20) showed differential effects in their cytotoxic activity on the target cancer cell lines. Significant angiogenesis inhibitions of 16 and 19 were also observed.

Triterpene acids from Euscaphis japonica and assessment of their cytotoxic and anti-NO activities.

Six new triterpenoids, euscaphic acids G-L (1-6), along with nine known triterpene acids, and two known lignans were isolated from the ethanolic extract of the twigs of Euscaphis japonica. This is the first report concerning 1α,3ß-dihydroxy-12-oleanen-28-oic acid isolated from a natural source. The structures of the new compounds were established by spectroscopic analysis. The cytotoxic and anti-NO production activities for the isolates are also evaluated and discussed; compound 1, hederagenin (11), and arjunic acid (12) showed significant cytotoxicity against NCI-H460 cells, HT-29 cells, and CEM cells (IC50 = 1.64 ± 0.87, 2.11 ± 1.54, 1.73 ± 0.64 µM, respectively). Some of the isolated triterpenoids showed marginal inhibitions on NO production induced by LPS.

SRT1720 as an SIRT1 activator for alleviating paraquat-induced models of Parkinson's disease.

Epidemiological studies have linked herbicides and Parkinson's disease (PD), with the strongest associations resulting from long exposure durations. Paraquat (PQ), an herbicide, induces PD-like syndromes and has widely been accepted as a PD mimetic. Currently, there is still no cure to prevent the progression of PD, and the search for effective therapeutic ways is urgent. Recently, the impairing activity of sirtuins (SIRTs), such as SIRT1, may correlate with PD etiology. However, the nonspecificity of SIRT1 agonists has made the protective mechanisms against PD unclear and hampered the therapeutic application of SIRT1. Thus, this study investigated the protective mechanism and therapeutic potential of SRT1720, a more specific agonist for SIRT1 synthesized by Sirtris, in alleviating the toxicity of PQ-induced cellular and animal models of PD. Here we show that SRT1720 alleviates PQ-induced toxicity in cell and animal models. Genetic silencing and pharmacological inhibition of SIRT1 attenuated SRT1720's protection against PQ-induced toxicity. Moreover, SRT1720 not only attenuated PQ-induced increased oxidative stress and mitochondrial free radical formations but also decreased mitochondrial membrane potential. Furthermore, SRT1720 reversed PQ-induced decreased PGC-1α levels and mitochondrial biogenesis. Although PQ and SRT1720 elevated NRF2 and antioxidative enzyme levels, only PQ decreased antioxidative enzyme activity but not SRT1720. NRF2 and PGC-1α silencing attenuated SRT1720 protection against PQ-induced toxicity. SRT1720 targeted SIRT1 and activated downstream PGC-1α and NRF2 signalings to prevent PQ-induced toxicity involving oxidative stress and mitochondrial dysfunction. Thus, SRT1720 might have therapeutic potential in preventing PD.

5-deoxyflavones with cytotoxic activity from Mimosa diplotricha.

Bioassay-guided isolation of Mimosa diplotricha led to the isolation of four new 5-deoxyflavones, diplotrins A-C (1-3) and diplotasin (4), together with 12 known flavonoids, flavonolignans, and triterpenoids. On the basis of spectroscopic evidence, compounds 1-4 were characterized as 2',5'-dihydroxy-3,7,8,4'-tetramethoxyflavone (1), 3'-hydroxy-3,7,8,4'-tetramethoxyflavone (2), 2'-hydroxy-7,4',5'-trimethoxyflavone (3), and 4-hydroxy-3,10,11-trimethoxyisochromeno-[4,3-b]-chromen-7(5H)-one (4). The cytotoxic effects of these isolated compounds were evaluated against the A549, AGS, HT-29, and PC3 human cancer cell lines. Compounds 2 and 5″-methoxyhydnocarpin-D (5) showed the most potent antiproliferative activity.

Triterpene glycosides and phenylpropane derivatives from Staurogyne concinnula possessing anti-angiogenic activity.

After anti-angiogenic activity screening, the potential n-butanol layer partitioned from the ethanol extract of Staurogyne concinnula was conducted. Further purification by Diaion HP20 column and preparative HPLC chromatography, four undescribed triterpenoid saponin derivatives, along with the known baptisiasaponin I, and four known phenylpropanoid glycosides were isolated and characterized from n-butanol layer. The structures of isolated compounds were elucidated by ESI-MS, 1D, and 2D MNR data. Biological evaluation revealed that baptisiasaponin I possessed significant anti-angiogenic effects (IC50 4.0 ± 0.2 µM). Further mechanism of action of baptisiasaponin I by inhibition of integrin/FAK/paxillin signaling pathway and its downstream effectors as MMP2 and MMP9 are also presented.

Huang Lian Jie Du Tang attenuates paraquat-induced mitophagy in human SH-SY5Y cells: A traditional decoction with a novel therapeutic potential in treating Parkinson's disease.

Huang Lian Jie Du Tang (HLJDT) is a traditional Chinese medical decoction for heat-fire clearing and detoxication. Theoretically, the cause of Parkinson's disease (PD) has been attributed to the dysregulations of internal wind, phlegm, fire, and stasis. Thus, HLJDT has been used to treat PD. However, the molecular mechanism is unknown. Besides, paraquat (PQ) as an herbicide has been known to impair midbrain dopaminergic neurons, resemblance to the pathology of PD. Thus, the molecular mechanism of HLJDT in treating PD and PQ-induced in vitro PD model was investigated in this study. Primarily, the dose-response of PQ (0.1∼1 mM)-induced neurotoxicity for 24 h was performed in the human neuroblastoma SH-SY5Y cells. The LD50 of PQ is around 0.3 mM and was applied throughout the following experiments. The neutral red assay was used to estimate cell viability. Co-transfection of the mitochondrial marker and proapoptotic factor genes were applied to measure the release of mitochondrial proapoptotic factors during PQ intoxication and HLJDT protection. The fluorescent dyes were used to detect mitochondrial membrane potential and free radical formation. Western blot and dot-blot analysis and immunocytochemistry were used to estimate the level of proteins related to apoptosis and mitophagy. PINK1 gene silencing was used to determine the significance of mitophagy during PQ intoxication. In this study, HLJDT attenuated PQ-induced apoptosis in SH-SY5Y cells. HLJDT reversed PQ-induced decreased mitochondrial membrane potential and suppressed PQ-induced increased cytosolic and mitochondrial free radical formations and mitochondrial proapoptotic factor releases. Furthermore, HLJDT mitigated PQ-induced increases in full-length PINK1, phosphorylations of Parkin and ubiquitin, mitochondrial translocation of phosphorylated Parkin, and mitophagy. PINK1 gene silencing attenuated PQ-induced neurotoxicity. Therefore, HLJDT attenuated PQ-induced cell death by regulating mitophagy.

A novel anti-tumor/anti-tumor-associated fibroblast/anti-mPEG tri-specific antibody to maximize the efficacy of mPEGylated nanomedicines against fibroblast-rich solid tumor.

The therapeutic efficacy of methoxypolyethylene glycol (mPEG)-coated nanomedicines in solid tumor treatment is hindered by tumor-associated fibroblasts (TAFs), which promote tumor progression and form physical barriers. We developed an anti-HER2/anti-FAP/anti-mPEG tri-specific antibody (TsAb) for one-step conversion of mPEG-coated liposomal doxorubicin (Lipo-Dox) to immunoliposomes, which simultaneously target HER2+ breast cancer cells and FAP+ TAFs. The non-covalent modification did not adversely alter the physical characteristics and stability of Lipo-Dox. The TsAb-Lipo-Dox exhibited specific targeting and enhanced cytotoxicity against mono- and co-cultured HER2+ breast cancer cells and FAP+ TAFs, compared to bi-specific antibody (BsAb) modified or unmodified Lipo-Dox. An in vivo model of human breast tumor containing TAFs also revealed the improved tumor accumulation and therapeutic efficacy of TsAb-modified mPEGylated liposomes without signs of toxicity. Our data indicate that arming clinical mPEGylated nanomedicines with the TsAb is a feasible and applicable approach for overcoming the difficulties caused by TAFs in solid tumor treatment.

Cytotoxic Hexacyclic Triterpene Acids from Euscaphis japonica.

Six hexacyclic triterpene acids (1-6), named euscaphic acids A-F, and eight known triterpene acid compounds (7-14) were isolated from an ethanolic extract of twigs of Euscaphis japonica. Compounds 8 and 10 were isolated for the first time from a natural source. Triterpenes 1-6 possess hexacyclic skeletons with a 13α,27-cyclopropane ring. Structural elucidation of compounds 1-6 was established by spectroscopic methods, especially 2D NMR techniques ((1)H-(1)H COSY, HMQC, HMBC, and NOESY). Compounds 3, 4, and 14 showed significant cytotoxicity against different cancer cell lines [IC50 = 2.54 (NCI-H460), 3.61 (MCF-7), and 3.27 µM (CEM) for 3, 4, and 14, respectively].

Cytotoxic polyisoprenyl benzophenonoids from Garcinia subelliptica.

Six new polyisoprenyl benzophenonoids, (+/-)-garcinialiptone A (1, 2), garcinialiptone B (3), (-)-cycloxanthochymol (4), garcinialiptone C (5), and garcinialiptone D (6), along with three known compounds, xanthochymol (7), isoxanthochymol (8), and cycloxanthochymol (9), were isolated from the fruits of Garcinia subelliptica. The structures of 1-6 were elucidated by spectroscopic analysis. Biological evaluation showed that all compounds 1-9 exhibited cytotoxic activity against a small panel of human tumor cell lines (A549, DU145, KB, vincristine-resistant KB).

Adipocyte browning and resistance to obesity in mice is induced by expression of ATF3.

Billions of people have obesity-related metabolic syndromes such as diabetes and hyperlipidemia. Promoting the browning of white adipose tissue has been suggested as a potential strategy, but a drug still needs to be identified. Here, genetic deletion of activating transcription factor 3 (ATF3-/- ) in mice under a high-fat diet (HFD) resulted in obesity and insulin resistance, which was abrogated by virus-mediated ATF3 restoration. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate adipokine genes and induce adipocyte browning by suppressing the carbohydrate-responsive element-binding protein-stearoyl-CoA desaturase-1 axis. Furthermore, ST32da increased white adipose tissue browning and reduced lipogenesis in HFD-induced obese mice. The anti-obesity efficacy of oral ST32da administration was similar to that of the clinical drug orlistat. Our study identified the ATF3 inducer ST32da as a promising therapeutic drug for treating diet-induced obesity and related metabolic disorders.