Experimental and In Silico Analysis of Cordycepin and its Derivatives as Endometrial Cancer Treatment

The aim of this study was to investigate the inhibition effects of cordycepin and its derivatives on endometrial cancer cell growth. Cytotoxicity MTT assays, clonogenic assays, and flow cytometry were used to observe the effects on apoptosis and regulation of the cell cycle of Ishikawa cells under various concentrations of cordycepin, cisplatin, and combinations of the two. Validated in silico docking simulations were performed on 31 cordycepin derivatives against adenosine deaminase (ADA) to predict their binding affinities and hence their potential tendency to be metabolized by ADA. Cordycepin has a significant dose-dependent inhibitory effect on cell proliferation. The combination of cordycepin and cisplatin produced greater inhibition effects than did cordycepin alone. Apoptosis investigations confirmed the ability of cordycepin to induce the apoptosis of Ishikawa cells. The in silico results indicate that compound MRS5698 is least metabolized by ADA and has acceptable drug likeness and safety profiles. This is the first study to confirm the cytotoxic effects of cordycepin on endometrial cancer cells. This study also identified cordycepin derivatives with promising pharmacological and pharmacokinetic properties for further investigation in the development of new treatments for endometrial cancer     

Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt

Cordycepin (3'-deoxyadenosine), a major bioactive compound of Cordyceps militaris, has many pharmacological actions, such as anti-inflammatory and anticancer activities. In this study, the relationship between inhibition of cell motility and anti-invasive activity by cordycepin in LNCaP human prostate carcinoma cells was investigated. Within the concentration range that was not cytotoxic, cordycepin time-dependently inhibited cell motility and invasiveness of LNCaP cells. The inhibitory effects of cordycepin on cell invasiveness were associated with tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting indicated that cordycepin decreases levels of claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, cordycepin inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, and simultaneously increased levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in LNCaP cells. These findings suggest that cordycepin inhibits the migration and invasion of LNCaP cells by downregulating the activity of TJs and MMPs, possibly in association with suppression of Akt activation.     

Cordycepin inhibits the proliferation and progression of NPC by targeting the MAPK/ERK and β-catenin pathways

Cordycepin is an extract from the Cordyceps genus of ascomycete fungi. In the present study, the anticancer potential of cordycepin against nasopharyngeal carcinoma (NPC), and the potential underlying mechanisms, were investigated. Using Cell Counting Kit 8, wound-healing and Transwell assays, cordycepin was found to reduce the viability and inhibit the migration of C666-1 cells in a dose-dependent manner. In addition, in colony formation assays, co-treatment with cordycepin and cisplatin inhibited the proliferation of C666-1 cells. Furthermore, RNA sequencing analysis identified 72 significantly differentially expressed genes and different signaling pathways that may be regulated by cordycepin. After treatment with cordycepin, the expression levels of ERK1/2, phosphorylated ERK1/2 and β-catenin were significantly downregulated. Therefore, cordycepin may be a novel candidate for NPC treatment or a co-treatment candidate with cisplatin in chemotherapy.     

The heparan sulfate mimetic PG545 interferes with Wnt/β-catenin signaling and significantly suppresses pancreatic tumorigenesis alone and in combination with gemcitabine

The heparan sulfate mimetic PG545 has been shown to exert anti-angiogenic and anti-metastatic activity in vitro and in vivo cancer models. Although much of this activity has been attributed to inhibition of heparanase and heparan sulfate-binding growth factors, it was hypothesized that PG545 may additionally disrupt Wnt signaling, an important pathway underlying the malignancy of pancreatic cancer. We show that PG545, by directly interacting with Wnt3a and Wnt7a, inhibits Wnt/β-catenin signaling leading to inhibition of proliferation in pancreatic tumor cell lines. Additionally, we demonstrate for the first time that the combination of PG545 with gemcitabine has strong synergistic effects on viability, motility and apoptosis induction in several pancreatic cell lines. In an orthotopic xenograft mouse model, combination of PG545 with gemcitabine efficiently inhibited tumor growth and metastasis compared to single treatment alone. Also, PG545 treatment alone decreased the levels of β-catenin and its downstream targets, cyclin D1, MMP-7 and VEGF which is consistent with our in vitro data. Collectively, our findings suggest that PG545 exerts anti-tumor activity by disrupting Wnt/β-catenin signaling and combination with gemcitabine should be considered as a novel therapeutic strategy for pancreatic cancer treatment.     

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer.We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer.RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1.Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.     

Different effects of LDH-A inhibition by oxamate in non-small cell lung cancer cells

Higher rate of glycolysis has been long observed in cancer cells, as a vital enzyme in glycolysis, lactate dehydrogenase A (LDH-A) has been shown with great potential as an anti-cancer target. Accumulating evidence indicates that inhibition of LDH-A induces apoptosis mediated by oxidative stress in cancer cells. To date, it''s still unclear that whether autophagy can be induced by LDH-A inhibition. Here, we investigated the effects of oxamate, one classic inhibitor of LDH-A in non-small cell lung cancer (NSCLC) cells as well as normal lung epithelial cells. The results showed that oxamate significantly suppressed the proliferation of NSCLC cells, while it exerted a much lower toxicity in normal cells. As previous studies reported, LDH-A inhibition resulted in ATP reduction and ROS (reactive oxygen species) burst in cancer cells, which lead to apoptosis and G₂/M arrest in H1395 cells. However, when being exposed to oxamate, A549 cells underwent autophagy as a protective mechanism against apoptosis. Furthermore, we found evidence that LDH-A inhibition induced G₀/G₁ arrest dependent on the activation of GSK-3β in A549 cells. Taken together, our results provide useful clues for targeting LDH-A in NSCLC treatment and shed light on the discovery of molecular predictors for the sensitivity of LDH-A inhibitors.     

The apoptotic effect of cordycepin on human OEC-M1 oral cancer cell line

Cordycepin (3′-deoxyadenosine), a pure compound of Cordyceps sinensis, has been illustrated with anti-tumor effects. In the present study, the apoptotic effect of cordycepin on OEC-M1, a human oral squamous cancer cell line, was investigated by morphological observations, cell viability assay, annexin V-FITC analysis and flow cytometry methods. Results demonstrated that the number of rounded-up cell increased as treatment duration of cordycepin (100 μM) increased from 3 to 48 h, and the plasma membrane blebbing could be observed after 12 h treatment. In cell viability assay, cell surviving rate significantly decreased as the dosage and duration of cordycepin treatment increased (P < 0.05). Moreover, phosphatidylserine flipping on cell membrane could be detected with 3, 6 and 12 h cordycepin treatment, which indicated an early apoptotic phenomenon. Furthermore, cell cycle studies illustrated that the percentage of G1 phase cell declined as the dosages of cordycepin increased (10 μM to 5 mM), while the percentages of G2M and subG1 phase cell increased (P < 0.05) in 12, 24 and 48 h cordycepin treatment. These results further confirmed the apoptotic event. In conclusion, cordycepin significantly induced cell apoptotsis in OEC-M1 human oral squamous cancer cells. Wei-Ciao Wu and Jenn-Ren Hsiao contributed equally.     

国产虫草素对Hela细胞MMP-9、TIMP-1及TIMP-1 mRNA表达的调节

目的：观察国产虫草素对人宫颈癌Hela细胞生长,MMP-9,TIMP-1及TIMP-1mRNA表达的影响。方法：采用MTr法观察不同浓度的虫草素溶液（0,1,2,4mg／ml）对Hela细胞增殖抑制作用。人MMP-9／TIMP-1ELISA试剂盒及RT—PCR技术检测经不同浓度虫草素溶液（0,1,2,4mg／ml）处理后不同时间点（12,24,48h）Hela细胞MMP-9／TIMP-1及TIMP-1 mRNA表达的影响。结果：国产虫草素以剂量依赖方式抑制Hela细胞生长。不同浓度虫草素处理不同时问的Hela细胞分泌MMP-9呈轻度剂量依赖抑制方式,但未见统计学差异（P〉0．05）。可上调TIMP-1及其mRNA的表达,且呈明显剂量依赖方式,统计学差异显著（P〈0．05）,尤其在24h TIMP-1 mRNA的表达及蛋白分泌达最高峰。结论：国产虫草素可以以剂量依赖方式抑制肿瘤细胞的生长;可以通过上调TIMP—1 mRNA及蛋白的表达发挥潜在的抗肿瘤细胞侵润转移的作用,为国产新型抗肿瘤药物的临床研发及应用提供实验室依据。     

MicroRNA-17-5p-activated Wnt/β-catenin pathway contributes to the progression of liver fibrosis

Aberrant Wnt/β-catenin pathway contributes to the development of liver fibrosis. MicroRNAs (MiRNAs) are found to act as regulators of the activation of hepatic stellate cell (HSC) in liver fibrosis. However, whether miRNAs activate Wnt/β-catenin pathway in activated HSCs during liver fibrosis is largely unknown. In this study, we found that Salvianolic acid B (Sal B) treatment significantly inhibited liver fibrosis in CCl₄-treated rats, HSC-T6 cells and rat primary HSCs, resulting in the suppression of type I collagen and alpha-smooth muscle actin. Also, Sal B suppressed HSC activation and cell proliferation in vitro. Interestingly, Sal B treatment induced the inactivation of Wnt/β-catenin pathway, with an increase in P-β-catenin and Wnt inhibitory factor 1 (WIF1). We demonstrated that the anti-fibrotic effects caused by Sal B were, at least in part, via WIF1. Moreover, our study revealed that miR-17-5p was reduced in vivo and in vitro after Sal B treatment. As confirmed by luciferase activity assays, WIF1 was a direct target of miR-17-5p. Notably, the suppression of HSCs induced by Sal B was almost inhibited by miR-17-5p mimics. Collectively, we demonstrated that miR-17-5p activates Wnt/β-catenin pathway to result in HSC activation through inhibiting WIF1 expression.     

Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis

Background

To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target.

Aim

To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells.

Methods

Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5–200 μM) for 24–96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot).

Results

In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability.

Conclusions

Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.     

Effects of salidroside on glioma formation and growth inhibition together with improvement of tumor microenvironment

Objective：To test the effects of salidroside on formation and growth of glioma together with tumor microenvironment.Methods：Salidroside extracted from Rhodiola rosea was purified and treated on human glioma cells U251 at the concentration of 20 μg/mL.3-（4,5-dimethylthiazol-2-yl）-2,5-dephenyltetrazolium bromide （MTT） assay for cytotoxicity and flow cytometry （FCM） for cell cycle analysis were performed.Then for in vivo study,xenotransplantation tumor model in nude mice was generated and treated with salidroside at the concentration of 50 mg/kg.d for totally 20 d.Body weight and tumor size were detected every 2 d after the treatment.The levels of 8-isoprostane,superoxide dismutase （SOD） and malondialdehyde （MDA）,special markers for oxidative stress,were detected while immunofluoresence staining was performed for astrocyte detection.Results：For in vitro study,salidroside could decrease the viability of human glioma cells U251 and the growth of U251 cells at G0/G1 checkpoint during the cell cycle.For in vivo study,salidroside could also inhibit the growth of human glioma tissue in nude mice.The body weight of these nude mice treated with salidroside did not decrease as quickly as control group.In the tumor xenotransplantation nude mice model,mice were found of inhibition of oxidative stress by detection of biomarkers.Furthermore,overgrowth of astrocytes due to the stimulation of oxidative stress in the cortex of brain was inhibited after the treatment of salidroside.Conclusions：Salidroside could inhibit the formation and growth of glioma both in vivo and in vitro and improve the tumor microenvironment via inhibition of oxidative stress and astrocytes.     

Fasting induces anti-Warburg effect that increases respiration but reduces ATP-synthesis to promote apoptosis in colon cancer models

Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called “Warburg effect” whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O₂ consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex II-dependent O₂ consumption, elevated oxidative stress and apoptosis.These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.     

Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer

Metastatic lung cancer is incurable and a leading cause of cancer death in the United States. However, the molecular mechanism by which lung cancer cells invade other tissues has remained unclear. We previously identified fibulin-5, an extracellular matrix protein, as a frequently silenced gene in lung cancer and a suppressor of cell invasion. In this study, we found fibulin-5 functions by inhibiting the Wnt/β-catenin pathway. The Cancer Genome Atlas (TCGA) datasets show a strong association between loss of fibulin-5 expression and poor outcomes of lung cancer patients, and also activation of the Wnt target genes MMP-7 and c-Myc. Fibulin-5 impedes Wnt/β-catenin signaling by inhibiting extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase-3 β (GSK3β), which downregulates β-catenin and prevents its nuclear accumulation, leading to suppression of MMP-7 and c-Myc expression. These effects of fibulin-5 are mediated by its amino-terminal integrin-binding RGD motif. Fibulin-5 also blocks Wnt/β-catenin signaling in vivo in H460 metastasis and H1299 tumor models. Furthermore, knockdown of β-catenin suppresses metastasis of H460 tumors, while knockdown of GSK3β promotes metastasis of fibulin-5-expressing H1752 tumors. Together, our results suggest that fibulin-5 functions as a metastasis suppressor in lung cancer by modulating tumor microenvironment to suppress Wnt/β-catenin signaling.