Corrigendum: A novel Chinese herbal and corresponding chemical formula for cancer treatment by targeting tumor maintenance, progression, and metastasis.

[This corrects the article DOI: 10.3389/fphar.2022.907826.].

The Effects of a Curcumin Derivative and Osimertinib on Fatty Acyl Metabolism and Mitochondrial Functions in HCC827 Cells and Tumors.

Drug combination therapy is a key approach in cancer treatments, aiming to improve therapeutic efficacy and overcome drug resistance. Evaluation of intracellular response in cancer cells to drug treatment may disclose the underlying mechanism of drug resistance. In this study, we aimed to investigate the effect of osimertinib, a tyrosine kinase inhibitor (TKI), and a curcumin derivative, 35d, on HCC827 cells and tumors by analyzing alterations in metabolome and related regulations. HCC827 tumor-bearing SCID mice and cultured HCC827 cells were separately examined. The treatment comprised four conditions: vehicle-only, 35d-only, osimertinib-only, and a combination of 35d and osimertinib. The treated tumors/cells were subsequently subjected to metabolomics profiling, fatty acyl analysis, mitochondrial potential measurement, and cell viability assay. Osimertinib induced changes in the ratio of short-chain (SC) to long-chain (LC) fatty acyls, particularly acylcarnitines (ACs), in both tumors and cells. Furthermore, 35d enhanced this effect by further lowering the SC/LC ratio of most ACs. Osimertinib and 35d also exerted detrimental effects on mitochondria through distinct mechanisms. Osimertinib upregulated the expression of carnitine palmitoyltransferase I (CPTI), while 35d induced the expression of heat shock protein 60 (HSP60). The alterations in ACs and CPTI were correlated with mitochondrial dysfunction and inhibited cell growth. Our results suggest that osimertinib and 35d disrupted the fatty acyl metabolism and induced mitochondrial stress in cancer cells. This study provides insights into the potential application of fatty acyl metabolism inhibitors, such as osimertinib or other TKIs, and mitochondrial stress inducers, such as curcumin derivatives, as combination therapy for cancer.

Docosahexaenoic Acid Alleviates Trimethylamine-N-oxide-mediated Impairment of Neovascularization in Human Endothelial Progenitor Cells.

BACKGROUND: Human endothelial progenitor cells (hEPCs), originating from hemangioblasts in bone marrow (BM), migrate into the blood circulation, differentiate into endothelial cells, and could act as an alternative tool for tissue regeneration. In addition, trimethylamine-N-oxide (TMAO), one of the gut microbiota metabolites, has been identified as an atherosclerosis risk factor. However, the deleterious effects of TMAO on the neovascularization of hEPCs have not been studied yet. RESULTS: Our results demonstrated that TMAO dose-dependently impaired human stem cell factor (SCF)-mediated neovascularization in hEPCs. The action of TMAO was through the inactivation of Akt/endothelial nitric oxide synthase (eNOS), MAPK/ERK signaling pathways, and an upregulation of microRNA (miR)-221. Docosahexaenoic acid (DHA) could effectively inhibit the cellular miR-221 level and induce the phosphorylation level of Akt/eNOS, MAPK/ERK signaling molecules, and neovascularization in hEPCs. DHA enhanced cellular amounts of reduced form glutathione (GSH) through an increased expression of the gamma-glutamylcysteine synthetase (γ-GCS) protein. CONCLUSIONS: TMAO could significantly inhibit SCF-mediated neovascularization, in part in association with an upregulation of miR-221 level, inactivation of Akt/eNOS and MAPK/ERK cascades, suppression of γ-GCS protein, and decreased levels of GSH and GSH/GSSG ratio. Furthermore, the DHA could alleviate the detrimental effects of TMAO and induce neovasculogenesis through suppression of miR-221 level, activation of Akt/eNOS and MAPK/ERK signaling cascades, increased expression of γ-GCS protein, and increment of cellular GSH level and GSH/GSSG ratio in hEPCs.

IRG1/Itaconate induces metabolic reprogramming to suppress ER-positive breast cancer cell growth.

Most breast cancers are estrogen receptor (ER)-positive, targeted by endocrine therapies, but chemoresistance remains a significant challenge in treating the disease. Altered intracellular metabolite has closely connected with the pathogenic process of breast cancer and drug resistance. Itaconate is an anti-inflammatory metabolite generated from converting cis-aconitate in the tricarboxylic acid (TCA) cycle by the immune response gene 1 (IRG1). However, the potential role of IRG1/Itaconate in the crosstalk of metabolic pathways and tumor development is currently unknown. We tested the hypothesis that IRG1/Itaconate controls metabolic homeostasis to modulate breast cancer cell growth. We showed that breast cancers harboring an IRG1 deletion displayed a worse prognosis than those without IRG1 deletion; approximately 70% of breast cancer with IRG1 deletion were ER-positive. There was no significant difference in the IRG1 copy number, mRNA, and protein levels between ER-positive and ER-negative breast cancer cell lines and breast tumors. Itaconate selectively inhibited ER-positive breast cancer cell growth via the blockade of DNA synthesis and the induction of apoptosis. Mechanistically, IRG1 overexpression led to decreased intermediate levels of glycolysis, the TCA cycle, and lipid metabolism to compromise the entire biomass and energy of the cell. Itaconate inhibited the enzymatic activity of succinate dehydrogenase (SDH) in the mitochondrial electron-transport chain, concomitant with reactive oxygen species (ROS) production and the decreased adenylate kinase (AK) activities, which, in turn, induced AMP-activated protein kinase (AMPK) activation to restore metabolic homeostasis. These results suggest a new regulatory pathway whereby IRG1/Itaconate controls metabolic homeostasis in ER-positive breast cancer cells, which may contribute to developing more efficacious therapeutic strategies for breast cancer.

H2S- and Redox-State-Mediated PTP1B S-Sulfhydration in Insulin Signaling.

Because hydrogen sulfide (H2S) is classified as a gaseous signaling molecule, protein S-sulfhydration is known to be one of the mechanisms by which H2S signals are conducted. PTP1B, a negative regulator in insulin signaling, has been found to be S-sulfhydrated at Cys215-SH to form Cys215-SSH in response to endoplasmic reticulum (ER) stress. Therefore, we aimed to understand the change in PTP1B S-sulfhydration and cellular redox homeostasis in response to insulin stimulation. We demonstrated a feasible PEG-switch method to determine the levels of PTP1B S-sulfhydration. According to the results obtained from HEK293T and MDA-MB-231 cells, insulin induced a change in PTP1B S-sulfhydration that was similar to the change in Insulin receptor substrate 1 (IRS1) phosphorylation in both cell lines. However, insulin-induced PTP1B S-sulfhydration and IRS1 phosphorylation were only significantly affected by metformin in HEK293T cells. Insulin also induced an increase in reactive oxygen species (ROS) in both cell lines. However, the level of H2S, GSH, and GSSG was only significantly affected by insulin and metformin in HEK293T cells. HEK293T cells maintained high levels of H2S and cysteine, but low levels of GSSG and GSH in general compared to MDA-MB-231 cells. From these findings, we suggest that PTP1B activity is modulated by H2S and redox-regulated S-sulfhydration during insulin signaling.

METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.

BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive. RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α. CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.

A Novel Chinese Herbal and Corresponding Chemical Formula for Cancer Treatment by Targeting Tumor Maintenance, Progression, and Metastasis.

We characterized a so-called "heirloom recipe" Chinese herbal formula (temporarily named Formula X) that contains five Chinese medical botanical drugs, Huang-Lian (Coptis chinensis Franch. [Ranunculaceae]), Huang-Qin (Scutellaria baicalensis Georgi [Lamiaceae]), Bai-Wei (Vincetoxicum atratum (Bunge) C. Morren and Decne. [Apocynaceae]), E-Zhu (Curcuma aromatica Salisb. [Zingiberaceae]) and Bai-Zhu (Atractylodes macrocephala Koidz. [Asteraceae]). Formula X inhibited the growth of various cancer cells and decreased the expression levels of a panel of proteins, including CD133, Myc, PD-L1, and Slug, in cancer cells. We further found that the inhibition of growth and protein expression were exerted by Huang-Lian, Huang-Qin, and Bai-Wei (formula HHB), which exhibited the same biological effects as those of Formula X. Furthermore, we selected three active chemicals, berberine, baicalin, and saponin from Huang-Lian, Huang-Qin, and Bai-Wei, respectively, to produce a chemical formulation (formula BBS), which exhibited similar effects on cell growth and protein expression as those induced by formula HHB. Both the formulae HHB and BBS suppressed tumor growth in an animal study. Moreover, they decreased the protein levels of Myc and PD-L1 in tumor cells in vivo. In summary, we established a novel Chinese herbal formula and a chemical formula that targeted three important processes, tumor maintenance (tumor stem cells), progression, and metastasis, and that influenced the response of tumors to host immunosuppression, for the potentially effective treatment of cancer patients.

Synthesis and Characterization of the Ethylene-Carbonate-Linked L-Valine Derivatives of 4,4-Dimethylcurcumin with Potential Anticancer Activities.

Natural phenolic products from herbal medicines and dietary plants constitute the main source of lead compounds for the development of the new drug. 4,4-Dimethylcurcumin (DMCU) is a synthetic curcumin derivative and exhibits anticancer activities against breast, colon, lung, and liver cancers. However, further development of DMCU is limited by unfavorable compound properties such as very low aqueous solubility and moderate stability. To increase its solubility, we installed either or both of the ethylene-carbonate-linked L-valine side chains to DMCU phenolic groups and produced targeted 1-trifluoroacetic acid (1-TFA) and 2-trifluoroacetic acid (2-TFA) derivatives. The terminus L-valine of ethylene-carbonate-linked side chain is known to be a L-type amino acid transporter 1 (LAT1) recognition element and therefore, these two derivatives were expected to readily enter into LAT1-expressing cancer cells. In practice, 1-TFA or 2-TFA were synthesized from DMCU in four steps with 34-48% overall yield. Based on the corresponding LC-MS analysis, water solubility of DMCU, 1-TFA, and 2-TFA at room temperature (25 ± 1 °C) were 0.018, 249.7, and 375.8 mg/mL, respectively, indicating >10,000-fold higher solubility of 1-TFA and 2-TFA than DMCU. Importantly, anti-proliferative assay demonstrated that 2-TFA is a potent anti-cancer agent against LAT1-expressing lung cancer cells NCI-H460, NCI-H358, and A549 cells due to its high intracellular uptake compared to DMCU and 1-TFA. In this study, we logically designed and synthesized the targeted compounds, established the LC-MS analytical methods for evaluations of drug solubility and intracellular uptake levels, and showed improved solubility and anti-cancer activities of 2-TFA. Our results provide a strategical direction for the future development of curcuminoid-like phenolic compounds.

Docosahexaenoic Acid Inhibits Cell Proliferation through a Suppression of c-Myc Protein in Pancreatic Ductal Adenocarcinoma Cells.

Treatment of pancreatic cancer by inhibiting the aberrant activation of the survival signaling pathways has received considerable attention. We investigated the probable action of DHA on the suppression of cell proliferation in human pancreatic ductal adenocarcinoma (PDAC) cells. Our results demonstrated that DHA dose-dependently inhibited cell proliferation through an induction of cell cycle arrest in human PDAC cells. DHA suppressed the expression of phosphorylated-Rb (p-Rb), cyclin D1, cyclin E, cyclin A, E2F1 and c-Myc proteins. Blocking the activation of STAT3 signaling pathway led to an inactivation of CAMKII and increased phosphorylation of c-Myc (T58) protein accompanied with decreased expression of c-Myc protein. Treatment of DHA effectively inhibited cell survival through decreased phosphorylation levels of EGFR, STAT3 and CAMKII proteins. The mechanisms of action were associated with increased phosphorylation levels of c-Myc (T58) and instability of c-Myc proteins. DHA inhibited cell survival through an increased GSSG/GSH ratio and oxidative stress level in HPAF-II cells. DHA induced cell apoptosis through increased expression of Bax, c-caspase 3 and c-PARP proteins in HPAF-II cells. Moreover, treatment of DHA significantly inhibited nucleotide synthesis. In conclusion, DHA might significantly suppress the proliferation of PDAC cells and therefore have potential as an anti-cancer therapeutic agent.

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD.

Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.

Development of Novel Rhodacyanine-Based Heat Shock Protein 70 Inhibitors.

BACKGROUND: A growing body of evidence suggests that Hsp70, which is overexpressed in human breast tumors, plays a role in tumorigenesis and tumor progression in breast cancer as well as in its aggressive phenotypes. Hsp70 constitutes a potential therapeutic target in the treatment of this disease. METHODS: We developed a new series of rhodacyanine-based Hsp70 inhibitors, represented by compounds 1 and 6, in which the cationic pyridin-1-ium or thiazol-3-ium ring of existing Hsp70 inhibitors (e.g., JG-40 and JG-98) was replaced by a corresponding benzo- fused N-heterocycle. RESULTS: Several lines of evidence suggest that these benzo-fused derivatives may exert their antitumor activities, in part, by targeting Hsp70. These putative inhibitors displayed differential antiproliferative efficacy against breast cancer cells (IC50 as low as 0.25 µM) versus nontumorigenic MCF-10A breast epithelial cells (IC50 ≥ 5 µM). This was correlated with the corresponding Hsp70 expression levels. Using a protein refolding assay, we confirmed that these agents effectively inhibited the chaperone activity of Hsp70. Moreover, these inhibitors effectively suppressed the expression of well-known oncogenic client proteins of Hsp70's, including FoxM1, HuR, and Akt, which paralleled their antiproliferative efficacy. Supporting the established role of Hsp70 in regulating protein refolding, these derivatives induced autophagy, as manifested by the conversion of LC3B-I to LC3B-II. Notably, these putative Hsp70 inhibitors did not cause a compensatory elevation in Hsp90 expression, contrasting with the previously reported effects of Hsp90 inhibitors on Hsp70 upregulation. CONCLUSION: Together with the finding that compounds 1 and 6 showed improved microsomal stability, these results suggest the translational potential of these putative Hsp70 inhibitors to foster new strategies for cancer therapy. However, whether these benzo-fused rhodacyanines act on kinases or other targets remains unclear. It is currently under investigation.

Docosahexaenoic acid inhibits the proliferation of Kras/TP53 double mutant pancreatic ductal adenocarcinoma cells through modulation of glutathione level and suppression of nucleotide synthesis.

The treatment of cancer cells obtained by blocking cellular metabolism has received a lot of attention recently. Previous studies have demonstrated that Kras mutation-mediated abnormal glucose metabolism would lead to an aberrant cell proliferation in human pancreatic ductal adenocarcinoma (PDAC) cells. Previous literature has suggested that consumption of fish oil is associated with lower risk of pancreatic cancer. In this study, we investigated the anti-cancer effects of docosahexaenoic acid (DHA) in human PDAC cells in vitro and in vivo. Omega-3 polyunsaturated fatty acids (PUFAs) such as DHA and eicosapentaenoic acid (EPA) significantly inhibited the proliferation of human PDAC cells. The actions of DHA were evaluated through an induction of cell cycle arrest at G1 phase and noticed a decreased expression of cyclin A, cyclin E and cyclin B proteins in HPAF-II cells. Moreover, it was found that co-treatment of DHA and gemcitabine (GEM) effectively induced oxidative stress and cell death in HPAF-II cells. Interestingly, DHA leads to an increased oxidative glutathione /reduced glutathione (GSSG/GSH) ratio and induced cell apoptosis in HPAF-II cells. The findings in the study showed that supplementation of GSH or N-Acetyl Cysteine (NAC) could reverse DHA-mediated cell death in HPAF-II cells. Additionally, DHA significantly increased cellular level of cysteine, cellular NADP/NADPH ratio and the expression of cystathionase (CTH) and SLCA11/xCT antiporter proteins in HPAF-II cells. The action of DHA was, in part, associated with the inactivation of STAT3 cascade in HPAF-II cells. Treatment with xCT inhibitors, such as erastin or sulfasalazine (SSZ), inhibited the cell survival ability in DHA-treated HPAF-II cells. DHA also inhibited nucleotide synthesis in HPAF-II cells. It was demonstrated in a mouse-xenograft model that consumption of fish oil significantly inhibited the growth of pancreatic adenocarcinoma and decreased cellular nucleotide level in tumor tissues. Furthermore, fish oil consumption induced an increment of GSSG/GSH ratio, an upregulation of xCT and CTH proteins in tumor tissues. In conclusion, DHA significantly inhibited survival of PDAC cells both in vitro and in vivo through its recently identified novel mode of action, including an increment in the ratio of GSSG/GSH and NADP/NADPH respectively, and promoting reduction in the levels of nucleotide synthesis.

A systematic identification of anti-inflammatory active components derived from Mu Dan Pi and their applications in inflammatory bowel disease.

Mu Dan Pi (MDP), also known as Moutan Cortex Radicis, is a traditional Chinese medicine used to treat autoimmune diseases. However, the impact of MDP and its principal active compounds on inflammatory bowel disease (IBD) is uncertain. This study therefore systemically assessed the anti-inflammatory effects of MDP and its known active compounds in IBD. The anti-inflammatory activities of water extract and individual compounds were screened by NF-κB and interferon regulatory factor (IRF) reporter assays in THP-1 cells induced with either Toll-like receptor or retinoic acid inducible gene I/melanoma differentiation-associated gene 5 activators and further verified in bone marrow-derived macrophages. MDP water extract significantly inhibited the activation of NF-κB and IRF reporters, downstream signaling pathways and the production of IL-6 and TNF-α, in a dose-dependent manner. Among 5 known active components identified from MDP (1,2,3,4,6-penta-O-galloyl-ß-d-glucose [PGG], gallic acid, methyl gallate, paeoniflorin, and paeonol), PGG was the most efficient at inhibiting both reporters (with an IC50 of 5-10 µM) and downregulating IL-6 and TNF-α. Both MDP powder for clinical use and MDP water extract, but not PGG, reduced colitis and pathological changes in mice. MDP and its water extract show promise as a novel therapy for IBD patients.

Electroacupuncture at Zusanli and at Neiguan characterized point specificity in the brain by metabolomic analysis.

Different point stimulations can induce brain activity in specific regions, and however whether these stimulations affect unique neurotransmitter transmission remains unknown. Therefore, we aimed to investigate the effect of point specificity to the brain by resolving the metabolite profiles. Eighteen Sprague-Dawley rats were randomly divided into three groups: (1) the sham group: sham acupuncture at Zusanli (ST36) without electric stimulation; (2) the Zusanli (ST36) group: electroacupuncture (EA) at ST36; and (3) the Neiguan (PC6) group: EA at PC6. Then, the metabolites from rat brain samples were measured by LC-ESI-MS. The results of a partial least squares discriminant analysis revealed the differences among the sham, ST36, and PC6 groups regarding the relative content of metabolites in the cerebral cortex, hippocampus, and hypothalamus. EA at PC6 resulted in downregulation of adenosine, adrenaline, γ-aminobutyric acid, glycine, and glutamate majorly in hippocampus, and then in cerebral cortex. Otherwise, EA at ST6 resulted in upregulation of adrenaline and arginine in hippocampus, and all stimulations showed barely change of identified neurotransmitters in hypothalamus. These differential metabolite and neurotransmitter profiles prove that brain areas can be modulated by point specificity and may provide a maneuver to understand more details of meridian.

Synthesis, Anticancer Activity, and Preliminary Pharmacokinetic Evaluation of 4,4-Disubstituted Curcuminoid 2,2-bis(Hydroxymethyl)Propionate Derivatives.

Compound 1 is a curcumin di-O-2,2-bis(hydroxymethyl)propionate that shows significant in vitro and in vivo inhibitory activity against MDA-MB-231 cells with eight to ten-fold higher potency than curcumin. Here, we modified the α-position (C-4 position) of the central 1,3-diketone moiety of 1 with polar or nonpolar functional groups to afford a series of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)propionate derivatives and evaluated their anticancer activities. A clear structure-activity relationship of compound 1 derivatives focusing on the functional groups at the C-4 position was established based on their anti-proliferative effects against the MDA-MB-231 and HCT-116 cell lines. Compounds 2-6 are 4,4-dimethylated, 4,4-diethylated, 4,4-dibenzylated, 4,4-dipropargylated and 4,4-diallylated compound 1, respectively. Compounds 2m-6m, the ester hydrolysis products of compounds 2-6, respectively, were synthesized and assessed for anticancer activity. Among all compound 1 derivatives, compound 2 emerged as a potential chemotherapeutic agent for colon cancer due to the promising in vivo anti-proliferative activities of 2 (IC50 = 3.10 ± 0.29 µM) and its ester hydrolysis product 2m (IC50 = 2.17 ± 0.16 µM) against HCT-116. The preliminary pharmacokinetic evaluation of 2 implied that 2 and 2m are main contributors to the in vivo efficacy. Compound 2 was further evaluated in an animal study using HCT-116 colon tumor xenograft bearing nude mice. The results revealed a dose-dependent efficacy that led to tumor volume reductions of 27%, 45%, and 60% at 50, 100, and 150 mg/kg doses, respectively. The established structure-activity relationship and pharmacokinetic outcomes of 2 is the guidance for future development of 4,4-disubstituted curcuminoid 2,2-bis(hydroxymethyl)- propionate derivatives as anticancer drug candidates.

Aqueous extracts of Paeonia suffruticosa modulates mitochondrial proteostasis by reactive oxygen species-induced endoplasmic reticulum stress in pancreatic cancer cells.

BACKGROUND: Pancreatic cancer (PC) remains the leading cause of cancer mortality, with limited therapeutic targets, and alterations in endoplasmic reticulum (ER)-related proteostasis may be a potential target for therapy. The root bark of Paeonia suffruticosa has been shown to inhibit cancer growth and metastasis, although its impact on PC is unknown. PURPOSE: To ascertain the anti-cancer effects of P. suffruticosa on oncogenic functions of PC and determine the detailed molecular mechanisms. STUDY DESIGN: Efficacy assessment of extracts, in vitro using PC cells as a model system and in vivo in mouse xenograft tumors. METHODS: P. suffruticosa aqueous extracts (PS) were prepared and assessed using liquid chromatography-tandem mass spectrometry. Cell viability, proteins, and cell components were measured using MTT assay, western blotting, and immunofluorescence. Cell apoptosis, cell cycle, and migration were assessed using colorimetric assays, fluorescence activated cell sorting, and transwell migration. Reactive oxygen species (ROS) were evaluated with a commercial 2'-7'-dichlorofluorescin diacetate kit. For the xenograft model, AsPC1 cells were inoculated subcutaneously into immunocompromised mice and PS (oral) was administered over 3 weeks with or without gemcitabine (GEM, intraperitoneal), a first-line advanced/metastatic PC therapy. RESULTS: PS stimulated ER stress and affected mitochondrial membrane potential to increase autophagosome numbers and block their degradation, followed by autophagy induction and finally cell apoptosis. Additionally, PS-mediated proteostasis impairment resulted in altered dynamics of the actin cytoskeleton, cell motility impairment, and cell cycle progression inhibition. Conversely, a ROS scavenger partially reversed PS-mediated degradation of peptidyl-prolyl cis-trans isomerase B (PPIB), an ER protein important for protein folding, suggesting that ROS generation by PS may be the upstream of PS-triggering of mitophagy and final cell apoptosis. Nevertheless, oral administration of PS, alone or in combination with GEM, delayed tumor growth in a xenograft model without affecting body weight. CONCLUSION: These findings indicate that PS may constitute a potential new alternative or complementary medicine for PC.

Quantitative and comparative liquid chromatography-electrospray ionization-mass spectrometry analyses of hydrogen sulfide and thiol metabolites derivaitized with 2-iodoacetanilide isotopologues.

Hydrogen sulfide (H2S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H2S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H2S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). 13C6-Labeled and label-free 2-IAN efficiently react with H2S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of H2S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13C6-labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 µM linear ranges for DL-homocysteine and glutathione and 1 nM-1 µM linear ranges for l-cysteine and H2S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13C6-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis.

Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives.

Phenyl vinyl sulfone (PVS) and phenyl vinyl sulfonate (PVSN) inactivate protein tyrosine phosphatases (PTPs) by mimicking the phosphotyrosine structure and providing a Michael addition acceptor for the active-site cysteine residue of PTPs, thus forming covalent adducts between PVS (or PVSN) and PTPs. We developed a specific antiserum against PVS. This antiserum can be used in general antibody-based assays such as immunoblotting, immunofluorescence staining, and immunoprecipitation. Target identification through immunoprecipitation and mass spectrometry analysis reveals potential targets of PVS, mostly proteins with reactive cysteine residues or low-pKa cysteine residues that are prone to reversible redox modifications. Target identification of PVSN has been conducted because the anti-PVS antiserum can also recognize PVSN. Among the targets, protein arginine methyltransferase 1 (PRMT1), inosine-5'-monophosphate dehydrogenase 1, vimentin, and glutathione reductase (GR) were further confirmed by immunoprecipitation followed by immunoblotting. In addition, PVSN and Bay11-7082 inhibited GR activity, and PVS, PVSN, and Bay 11-7082 inhibited PRMT1 activity in in vitro assays. In addition, treatment of PVSN, Bay11-7082, or Bay 11-7085 in cultured HeLa cells can cause the quick decline in the levels of protein asymmetric dimethylarginine. These results indicate that the similar moiety among PVS, PVSN, Bay 11-7082, and Bay 11-7085 can be the key structure of lead compounds of PRMT1. Therefore, we expect to use this approach in the identification of potential targets of other covalent drugs.

Enrichment of Metabolite-Binding Proteins by Affinity Elution in Tandem Hydrophobic Interaction Chromatography (AETHIC) Reveals RKIP Regulating ERK Signaling in an ATP-Dependent Manner.

To elucidate the molecular mechanisms underlying the action of bioactive compounds such as metabolites, identification of their binding targets is essential. However, available techniques for enriching metabolite-binding proteins are practically restrained by special equipment requirements and laborious efforts. Here we have developed a novel method, affinity elution in tandem hydrophobic interaction chromatography (AETHIC), which enables enrichment of metabolite-binding proteins from a crude tissue extract. AETHIC constitutes two major steps, protein fractionation and affinity elution. The basic strategy of AETHIC uses a series of HIC matrices encompassing aliphatic chains of different length and thus provides a wide range of hydrophobicity for interactions with most proteins. Thereafter, target proteins are eluted selectively by a given ligand. As our first proof-of-principle, we demonstrated that AETHIC was able to enrich ATP-binding proteins from porcine brain extract. In addition, we have demonstrated that raf kinase inhibitory protein (RKIP) is an ATP-binding protein and ATP attenuates the interaction between RKIP and Raf-1. In parallel, short-term ATP depletion in cultured HEK293 cells augments interaction between RKIP and Raf-1, resulting in decreased activation of the downstream ERK signaling. Therefore, the ATP-binding function renders RKIP's inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels affect the propagation of cellular signaling. Taken together, the enclosed results advocate the potential of AETHIC in the study of metabolite-protein interactions.

Post-staining electroblotting for efficient and reliable peptide blotting.

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.