Cordycepin Enhanced Therapeutic Potential of Gemcitabine against Cholangiocarcinoma via Downregulating Cancer Stem-Like Properties.

Cordycepin, a valuable bioactive component isolated from Cordyceps militaris, has been reported to possess anti-cancer potential and the property to enhance the effects of chemotherapeutic agents in various types of cancers. However, the ability of cordycepin to chemosensitize cholangiocarcinoma (CCA) cells to gemcitabine has not yet been evaluated. The current study was performed to evaluate the above, and the mechanisms associated with it. The study analyzed the effects of cordycepin in combination with gemcitabine on the cancer stem-like properties of the CCA SNU478 cell line, including its anti-apoptotic, migratory, and antioxidant effects. In addition, the combination of cordycepin and gemcitabine was evaluated in the CCA xenograft model. The cordycepin treatment significantly decreased SNU478 cell viability and, in combination with gemcitabine, additively reduced cell viability. The cordycepin and gemcitabine co-treatment significantly increased the Annexin V+ population and downregulated B-cell lymphoma 2 (Bcl-2) expression, suggesting that the decreased cell viability in the cordycepin+gemcitabine group may result from an increase in apoptotic death. In addition, the cordycepin and gemcitabine co-treatment significantly reduced the migratory ability of SNU478 cells in the wound healing and trans-well migration assays. It was observed that the cordycepin and gemcitabine cotreatment reduced the CD44highCD133high population in SNU478 cells and the expression level of sex determining region Y-box 2 (Sox-2), indicating the downregulation of the cancer stem-like population. Cordycepin also enhanced oxidative damage mediated by gemcitabine in MitoSOX staining associated with the upregulated Kelch like ECH Associated Protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) expression ratio. In the SNU478 xenograft model, co-administration of cordycepin and gemcitabine additively delayed tumor growth. These results indicate that cordycepin potentiates the chemotherapeutic property of gemcitabine against CCA, which results from the downregulation of its cancer-stem-like properties. Hence, the combination therapy of cordycepin and gemcitabine may be a promising therapeutic strategy in the treatment of CCA.

Identification of Crocetin as a Dual Agonist of GPR40 and GPR120 Responsible for the Antidiabetic Effect of Saffron.

Crocin, a glycoside of crocetin, has been known as the principal component responsible for saffron's antidiabetic, anticancer, and anti-inflammatory effects. Crocetin, originating from the hydrolytic cleavage of crocin in biological systems, was subjected to ligand-based virtual screening in this investigation. Subsequent biochemical analysis unveiled crocetin, not crocin, as a novel dual GPR40 and GPR120 agonist, demonstrating a marked preference for GPR40 and GPR120 over peroxisome proliferator-activated receptors (PPAR)γ. This compound notably enhanced insulin and GLP-1 secretion from pancreatic ß-cells and intestinal neuroendocrine cells, respectively, presenting a dual mechanism of action in glucose-lowering effects. Docking simulations showed that crocetin emulates the binding characteristics of natural ligands through hydrogen bonds and hydrophobic interactions, whereas crocin's hindered fit within the binding pocket is attributed to steric constraints. Collectively, for the first time, this study unveils crocetin as the true active component of saffron, functioning as a GPR40/120 agonist with potential implications in antidiabetic interventions.

Oxygen consumption rate to evaluate mitochondrial dysfunction and toxicity in cardiomyocytes.

The increase in the types and complexity of diseases has led to significant advances in diagnostic techniques and the availability of effective therapies. Recent studies have focused on the role of mitochondrial dysfunction in the pathogenesis of cardiovascular diseases (CVDs). Mitochondria are important organelles in cells that generate energy. Besides the production of adenosine triphosphate (ATP), the energy currency of cells, mitochondria are also involved in thermogenesis, control of intracellular calcium ions (Ca2+), apoptosis, regulation of reactive oxygen species (ROS), and inflammation. Mitochondrial dysfunction has been implicated in several diseases including cancer, diabetes, some genetic diseases, and neurogenerative and metabolic diseases. Furthermore, the cardiomyocytes of the heart are rich in mitochondria due to the large energy requirement for optimal cardiac function. One of the main causes of cardiac tissue injuries is believed to be mitochondrial dysfunction, which occurs via complicated pathways which have not yet been completely elucidated. There are various types of mitochondrial dysfunction including mitochondrial morphological change, unbalanced levels of substances to maintain mitochondria, mitochondrial damage by drugs, and mitochondrial deletion and synthesis errors. Most of mitochondrial dysfunctions are linked with symptoms and diseases, thus we focus on parts of mitochondrial dysfunction about fission and fusion in cardiomyocytes, and ways to understand the mechanism of cardiomyocyte damage by detecting oxygen consumption levels in the mitochondria.

Genetically engineered neural stem cells expressing cytosine deaminase and interferon-beta enhanced T cell-mediated antitumor immunity against gastric cancer in a humanized mouse model.

AIMS: Gastric cancer (GC) is an invasive, fatal disease with a poor prognosis. Gene-directed enzyme prodrug therapy via genetically engineered neural stem cells (GENSTECs) has been widely studied in various malignancies, such as breast, ovarian, and renal cancer. In this study, the human neural stem cells expressing cytosine deaminase and interferon beta (HB1.F3.CD.IFN-ß) cells were applied to convert non-toxic 5-fluorocytosine to cytotoxic 5-fluorouracil and secrete IFN-ß. MATERIALS AND METHODS: Human lymphokine-activated killer cells (LAKs) were generated by stimulating human peripheral blood mononuclear cells (PBMCs) by interleukin-2, and we evaluated the cytotoxic activity and migratory ability of LAKs co-cultured with GNESTECs or their conditioned media in vitro. A GC-bearing human immune system (HIS) mouse model was generated by transplanting human PBMCs followed by subcutaneous engraftment of MKN45 cells in NSG-B2m mice to evaluate the involvement of T cell-mediated anti-cancer immune activity of GENSTECs. KEY FINDINGS: In vitro studies showed the presence of HB1.F3.CD.IFN-ß cells facilitated the migration ability of LAKs to MKN45 cells and activated their cytotoxic potential. In MKN45-xenografted HIS mice, treatment with HB1.F3.CD.IFN-ß cells resulted in increased cytotoxic T lymphocyte (CTL) infiltration throughout the tumor, including the central area. Moreover, the group treated to HB1.F3.CD.IFN-ß showed increased granzyme B expression in the tumor, eventually enhancing the tumor-killing potential of CTLs and significantly delaying tumor growth. SIGNIFICANCE: These results indicate that the HB1.F3.CD.IFN-ß cells exert anti-cancer effects on GC by facilitating the T cell-mediated immune response, and GENSTECs are a promising therapeutic strategy for GC.

Methyl Syringate Stimulates Glucose Uptake by Inhibiting Protein Tyrosine Phosphatases Relevant to Insulin Resistance.

Several protein tyrosine phosphatases (PTPs), particularly PTPN1, PTPN2, PTPN6, PTPN9, PTPN11, PTPRS, and DUSP9, are involved in insulin resistance. Therefore, these PTPs could be promising targets for the treatment of type 2 diabetes. Our previous studies revealed that PTPN2 and PTPN6 are potential antidiabetic targets. Therefore, the identification of dual-targeting inhibitors of PTPN2 and PTPN6 could be a potential therapeutic strategy for the treatment or prevention of type 2 diabetes. In this study, we demonstrate that methyl syringate inhibits the catalytic activity of PTPN2 and PTPN6 in vitro, indicating that methyl syringate acts as a dual-targeting inhibitor of PTPN2 and PTPN6. Furthermore, methyl syringate treatment significantly increased glucose uptake in mature 3T3-L1 adipocytes. Additionally, methyl syringate markedly enhanced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in 3T3L1 adipocytes. Taken together, our results suggest that methyl syringate, a dual-targeting inhibitor of PTPN2 and PTPN6, is a promising therapeutic candidate for the treatment or prevention of type 2 diabetes.

Effects of CCN6 overexpression on the cell motility and activation of p38/bone morphogenetic protein signaling pathways in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types that is highly resistant to conventional treatments, such as chemotherapy and radiotherapy. As the demand for more effective therapeutics for PDAC treatment increases, various approaches have been studied to develop novel targets. The cellular communication network (CCN) family is a matricellular protein that modulates various cellular functions, including cell adhesion, proliferation, migration, and invasiveness. Despite this, little is known about the role of CCN6 in PDAC. The current study investigated the role of CCN6 in PDAC by generating CCN6-overexpressing PANC-1 cells (PANC-1-CCN6) by infecting lentivirus particles containing CCN6. PANC-1-CCN6 induces cell viability and tumorigenesis than PANC-1 cells with empty vector (control). The PANC-1-CCN6 formed more colonies, and the size of spheroids increased compared to the control. The upregulation of CCN6 enhances the expression of bone morphogenetic proteins (BMPs) genes and the upregulation of p38 mitogen-activated protein kinases (MAPKs). In PANC-1-CCN6 cells, the levels of N-cadherin, VEGF, and Snail expression were higher than the control, while E-cadherin expression was lower, which is associated with upregulation of epithelial-to-mesenchymal transition (EMT). Consistent with the changes in EMT-related proteins in PANC-1-CCN6, the migratory ability and invasiveness were enhanced in PANC-1-CCN6. Xenografted PANC-1-CCN6 in immunocompromised mice exhibited accelerated tumor growth than the control group. In immunohistochemistry (IHC), the PANC-1-CCN6 xenografted tumor showed an increased positive area of PCNA and Ki-67 than the control. These results suggest that CCN6 plays a tumorigenic role and induces the metastatic potential by the p38 MAPK and BMPs signaling pathways. Although the role of CCN6 has been introduced as an antitumor factor, there was evidence of CCN6 acting to cause tumorigenesis and invasion in PANC-1.

Next-Generation Antisense Oligonucleotide of TGF-ß2 Enhances T Cell-Mediated Anticancer Efficacy of Anti-PD-1 Therapy in a Humanized Mouse Model of Immune-Excluded Melanoma.

Anti-programmed death-1 (PD-1) immunotherapy is one of the most promising therapeutic interventions for treating various tumors, including lung cancer, bladder cancer, and melanoma. However, only a subset of patients responds to anti-PD-1 therapy due to complicated immune regulation in tumors and the evolution of resistance. In the current study, we investigate the potential of a novel transforming growth factor-beta2 (TGF-ß2) antisense oligonucleotide (ngTASO), as a combination therapy with an anti-PD-1 antibody in melanoma. This study was conducted in a melanoma-bearing human immune system mouse model that recapitulates immune-excluded phenotypes. We observed that the TGF-ß2 blockade by ngTASO in combination with PD-1 inhibition downregulated the tumor intrinsic ß-catenin, facilitated the infiltration of CD8+ cytotoxic lymphocytes (CTLs) in the tumor, and finally, enhanced the antitumor immune potentials and tumor growth delays. Blockade of TGF-ß2 combined with PD-1 inhibition also resulted in downregulating the ratio of regulatory T cells to CTLs in the peripheral blood and tumor, resulting in increased granzyme B expression. In addition, co-treatment of ngTASO and anti-PD-1 augmented the PD-L1 expression in tumors, which is associated with an improved response to anti-PD-1 immunotherapy. These results indicate that the combination of ngTASO and anti-PD-1 exerts an enhanced T cell-mediated antitumor immune potential. Hence, co-inhibition of TGF-ß2 and PD-1 is a potentially promising immunotherapeutic strategy for immune-excluded melanoma.

Establishment of a platform for measuring mitochondrial oxygen consumption rate for cardiac mitochondrial toxicity.

The heart has an abundance of mitochondria since cardiac muscles require copious amounts of energy for providing continuous blood through the circulatory system, thereby implying that myocardial function is largely reliant on mitochondrial energy. Thus, cardiomyocytes are susceptible to mitochondrial dysfunction and are likely targets of mitochondrial toxic drugs. Various methods have been developed to evaluate mitochondrial toxicity by evaluating toxicological mechanisms, but an optimized and standardized assay for cardiomyocytes remains unmet. We have therefore attempted to standardize the evaluation system for determining cardiac mitochondrial toxicity, using AC16 human and H9C2 rat cardiomyocytes. Three clinically administered drugs (acetaminophen, amiodarone, and valproic acid) and two anticancer drugs (doxorubicin and tamoxifen) which are reported to have mitochondrial effects, were applied in this study. The oxygen consumption rate (OCR), which directly reflects mitochondrial function, and changes in mRNA levels of mitochondrial respiratory complex I to complex V, were analyzed. Our results reveal that exposure to all five drugs results in a concentration-dependent decrease in the basal and maximal levels of OCR in AC16 cells and H9C2 cells. In particular, marked reduction in the OCR was observed after treatment with doxorubicin. The reduction in OCR after exposure to mitochondrial toxic drugs was found to be associated with reduced mRNA expression in the mitochondrial respiratory complexes, suggesting that the cardiac mitochondrial toxicity of drugs is majorly due to dysfunction of mitochondrial respiration. Based on the results of this study, we established and standardized a protocol to measure OCR in cardiomyocytes. We expect that this standardized evaluation system for mitochondrial toxicity can be applied as basic data for establishing a screening platform to evaluate cardiac mitochondrial toxicity of drugs, during the developmental stage of new drug discovery.

Network pharmacology and molecular docking approaches to elucidate the potential compounds and targets of Saeng-Ji-Hwang-Ko for treatment of type 2 diabetes mellitus.

BACKGROUND: Saeng-Ji-Hwang-Ko (SJHK) is a traditional Korean medicine formula derived from Donguibogam, a classic medical textbook, published in 1613. It is described as a general treatment for So-gal (wasting-thirst, ) known as type 2 diabetes mellitus (T2DM) in a modern clinical term. It is necessary to elucidate the potential compounds and targets of SJHK for T2DM treatment by conducting network pharmacology and molecular docking analyses. METHODS: Information about the chemical constituents of SJHK were collected, and druggable compounds were screened based on oral bioavailability and drug-likeness. Putative target genes of druggable compounds and T2DM-related genes were retrieved from public databases. A compound-target network was constructed to visualize the relationship between the druggable compounds in SJHK and common targets related to T2DM. The constructed network was further investigated through Protein-Protein Interaction, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway analyses, and molecular docking. RESULTS: Compound-target network analysis demonstrated that kaempferol, salicylic acid, estrone, and ß-sitosterol were key compounds of SJHK with PTGS2, ESR1, PRKAA2, PRKAB1, and CYP19A1 being its key targets. Estrogen signaling, AGE-RAGE signaling, insulin resistance, non-alcoholic fatty liver disease, and TNF signaling pathway were potential pathways involved in the effect of SJHK on T2DM. Molecular docking simulations revealed that estrone and ß-sitosterol had the strong binding energies for all the key target proteins. CONCLUSIONS: This study demonstrates that network pharmacology and molecular docking analyses help to better understand the potential key compounds and targets of SJHK for treating T2DM as a complementary medicine. SUMMARY: Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder caused by genetic and/or environmental factors. There has been a growing attention to new therapeutic approaches to treat T2DM using traditional medicine as a complementary treatment which is expected to have synergistic effects with few side effects. Saeng-Ji-Hwang-Ko (SJHK) is a traditional Korean medicine (TKM) formula derived from Donguibogam, a classic medical textbook, published in 1613. It is described as a general treatment for So-gal (wasting-thirst, ) known as type 2 diabetes mellitus (T2DM) in a modern clinical term. It is necessary to elucidate the potential compounds and targets of SJHK for T2DM treatment by conducting network pharmacology and molecular docking analyses. Compound-target network analysis demonstrated that kaempferol, salicylic acid, estrone, and ß-sitosterol were key compounds of SJHK with PTGS2, ESR1, PRKAA2, PRKAB1, and CYP19A1 being its key targets. Estrogen signaling, AGE-RAGE signaling, insulin resistance, non-alcoholic fatty liver disease, and TNF signaling pathway were potential pathways involved in the effect of SJHK on T2DM. Molecular docking evaluation revealed that estrone and ß-sitosterol had the highest binding energies for all key target proteins, suggesting potential key compounds of SJHK. Although additional future studies including further experimental and clinical validation are needed, this study demonstrates that SJHK has a great potential for treating T2DM as a complementary medicine.

Ethyl Gallate Dual-Targeting PTPN6 and PPARγ Shows Anti-Diabetic and Anti-Obese Effects.

The emergence of the high correlation between type 2 diabetes and obesity with complicated conditions has led to the coinage of the term "diabesity". AMP-activated protein kinase (AMPK) activators and peroxisome proliferator-activated receptor (PPARγ) antagonists have shown therapeutic activity for diabesity, respectively. Hence, the discovery of compounds that activate AMPK as well as antagonize PPARγ may lead to the discovery of novel therapeutic agents for diabesity. In this study, the knockdown of PTPN6 activated AMPK and suppressed adipogenesis in 3T3-L1 cells. By screening a library of 1033 natural products against PTPN6, we found ethyl gallate to be the most selective inhibitor of PTPN6 (Ki = 3.4 µM). Subsequent assay identified ethyl gallate as the best PPARγ antagonist (IC50 = 5.4 µM) among the hit compounds inhibiting PTPN6. Ethyl gallate upregulated glucose uptake and downregulated adipogenesis in 3T3-L1 cells as anticipated. These results strongly suggest that ethyl gallate, which targets both PTPN6 and PPARγ, is a potent therapeutic candidate to combat diabesity.

Nepetin Acts as a Multi-Targeting Inhibitor of Protein Tyrosine Phosphatases Relevant to Insulin Resistance.

Protein tyrosine phosphatases (PTPs) are essential modulators of signal transduction pathways and has been implicated in many human diseases such as cancer, diabetes, obesity, autoimmune disorders, and neurological diseases, indicating that PTPs are next-generation drug targets. Since PTPN1, PTPN2, and PTPN11 have been reported to be negative regulators of insulin action, the identification of PTP inhibitors may be an effective strategy to develop therapeutic agents for the treatment of type 2 diabetes. In this study, we observed for the first time that nepetin inhibits the catalytic activity of PTPN1, PTPN2, and PTPN11 in vitro, indicating that nepetin acts as a multi-targeting inhibitor of PTPN1, PTPN2, and PTPN11. Furthermore, treatment of mature 3T3-L1 adipocytes with 20 µM nepetin stimulates glucose uptake through AMPK activation. Taken together, our findings provide evidence that nepetin, a multi-targeting inhibitor of PTPN1, PTPN2, and PTPN11, could be a promising therapeutic candidate for the treatment of type 2 diabetes.

Polyphyllin D Shows Anticancer Effect through a Selective Inhibition of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP2).

Natural products have continued to offer tremendous opportunities for drug development, as they have long been used in traditional medicinal systems. SHP2 has served as an anticancer target. To identify novel SHP2 inhibitors with potential anticancer activity, we screened a library containing 658 natural products. Polyphyllin D was found to selectively inhibit SHP2 over SHP1, whereas two other identified compounds (echinocystic acid and oleanolic acid) demonstrated dual SHP1 and SHP2 inhibition. In a cell-based assay, polyphyllin D exhibited cytotoxicity in Jurkat cells, an acute lymphoma leukemia cell line, whereas the other two compounds were ineffective. Polyphyllin D also decreased the level of phosphorylated extracellular signal-regulated kinase (p-ERK), a proliferation marker in Jurkat cells. Furthermore, knockdown of protein tyrosine phosphatase (PTP)N6 (SHP1) or PTPN11 (SHP2) decreased p-ERK levels. However, concurrent knockdown of PTPN6 and PTPN11 in Jurkat cells recovered p-ERK levels. These results demonstrated that polyphyllin D has potential anticancer activity, which can be attributed to its selective inhibition of SHP2 over SHP1.

A FRET-Based Fluorescent Probe to Screen Anticancer Drugs, Inhibiting p73 Binding to MDM2.

The protein p73 acts as a transcription factor, resulting in tumour suppression. MDM2, an oncogenic protein, can negatively influence p73-mediated apoptosis by binding to p73 transactivation domains (TAD). Inhibition of the protein-protein interaction between p73 and oncogenic proteins is an attractive strategy for promoting p73-mediated apoptosis. Herein, we describe the use of a modified p73-TAD peptide for the FRET-based assay of the binding of p73-TAD to MDM2. The FRET probe, equipped with 1-naphthylamine (λex =330 nm, λem =445 nm), serves as a FRET acceptor, and the tryptophan of the protein acts as FRET donor (λex =280 nm, λem =340 nm). Sensitized emission from the FRET probe was observed upon excitation of the protein-FRET-probe complex at the excitation wavelength of Trp. Furthermore, addition of the MDM2 inhibitor Nutiln-3 drastically reduced the FRET signal, thus indicating that the FRET probe competes with Nutiln-3 for MDM2 binding. The developed FRET binding assay might be applicable in high-throughput screening of novel drugs that inhibit interactions between p73 and MDM2.

Identification of Vaccinia-H1 Related Phosphatase as an Anticancer Target for 1,2,3,4,6-O-Pentagalloylglucose.

Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia-H1 related phosphatase (VHR) has been shown to arrest at the G1-S and G2-M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anticancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR-inhibitory compounds, 1,2,3,4,6-O-pentagalloylglucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice, but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (Ki =53 nm) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl-2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.