Small Molecule Inhibitors of Wnt/β-Catenin/Lef-1 Signaling Induces Apoptosis in Chronic Lymphocytic Leukemia Cells In Vitro and In Vivo

Background

Lymphoid enhancer factor-1 (lef-1) is overexpressed in B-cell chronic lymphocytic leukemia (CLL) when compared with normal B cells and transcribes several genes implicated in the pathogenesis of CLL. We therefore hypothesize that antagonism of lef-1 might lead to killing of CLL cells. We used two small molecule inhibitors of Wnt/β-catenin/lef-1 signaling (CGP049090 and PKF115-584) to test our hypothesis.

Design and Methods

Enriched CLL cells and healthy B cells were used in this study. Small interfering RNA (siRNA)-mediated knockdown of lef-1 in primary CLL cells was done using nucleofection, and 50% lethal concentration (LC₅₀) of two small molecules was assessed using ATP-based cell viability assay. Apoptotic response was investigated in time course experiments with different apoptotic markers. Specificity of the small molecules was demonstrated by coimmunoprecipitation experiments for the lef-1/β-catenin interaction. In vivo studies were done in JVM-3 subcutaneous xenograft model.

Results

Inhibition of lef-1 by siRNA leads to increased apoptosis of CLL cells and inhibited proliferation of JVM-3 cell lines. The two small molecule inhibitors (CGP049090 and PKF115-584) efficiently kill CLL cells (LC₅₀<1 µM), whereas normal B cells were not significantly affected. Coimmunoprecipitation showed a selective disruption of β-catenin/lef-1 interaction. In vivo studies exhibited tumor inhibition of 69% with CGP049090 and 57% with PKF115-584 when compared with vehicle-treated controls, and the intervention was well tolerated.

Conclusions

We have demonstrated that targeting lef-1 is a new and selective therapeutic approach in CLL. CGP049090 or PKF115-584 may be attractive compounds for CLL and other malignancies that deserve further (pre)clinical evaluation.     

Dickkopfs and Wnt/β-catenin signalling in liver cancer

Liver cancer is the fifth and seventh most common cause of cancer in men and women, respectively. Wnt/β-catenin signalling has emerged as a critical player in both the development of normal liver as well as an oncogenic driver in hepatocellular carcinoma (HCC). Based on the current understanding, this article summarizes the possible mechanisms for the aberrant activation of this pathway with specific focus on HCC. Furthermore, we will discuss the role of dickkopfs (DKKs) in regulating Wnt/β-catenin signalling, which is poorly understood and understudied. DKKs are a family of secreted proteins that comprise at least four members, namely DKK1-DKK4, which act as inhibitors of Wnt/β-catenin signalling. Nevertheless, not all members antagonize Wnt/β-catenin signalling. Their functional significance in hepatocarcinogenesis remains to be further characterized for which these studies should provide new insights into the regulatory role of DKKs in Wnt/β-catenin signalling in hepatic carcinogenesis. Because of the important oncogenic roles, there are an increasing number of therapeutic molecules targeting β-catenin and the Wnt/β-catenin pathway for potential therapy of HCC.     

Wnt/β-catenin pathway in bone cancers

The Wnt signaling pathway regulates some of the crucial aspects of cellular processes. The beta-catenin dependent Wnt signaling (Wnt/β-catenin) pathway controls the expression of key developmental genes, and acts as an intracellular signal transducer. The association of Wnt/β-catenin pathway is often reported with different cancers. In this study, we have reviewed the association of Wnt/β-catenin pathway with bone cancers, focusing on carcinogenesis and therapeutic aspects. Wnt/β-catenin pathway is a highly complex and unique signaling pathway, which has ability to regulate gene expression, cell invasion, migration, proliferation, and differentiation for the initiation and progression of bone cancers, especially osteosarcoma. Association of Wnt/β-catenin pathway with chondrosarcoma, Ewing’s sarcoma and chondroma is also documented. Recently, targeting Wnt/β-catenin pathway has gained significant interests as a potential therapeutic application for the treatment of bone cancers. Small RNA technology to knockdown aberrant Wnt/β-catenin or inhibition of β-catenin expression by natural component has shown promising effects against bone cancers. Advances in understanding the mechanisms of Wnt signaling and new technologies have facilitated the discovery of agents that can target and regulate Wnt/β-catenin signaling pathway, and these may provide a basement for the innovative therapeutic approaches in the treatment of bone cancers.     

PLAGL1 Gene Demethylation Induced by Epigallocatechin Gallate Promotes Pheochromocytoma Cell Apoptosis Via Wnt/β-catenin Signaling Pathway

Objective: The aim of this study was to investigate the expression and the promoter methylation level of PLAGL1 gene and the mechanism of epigallocatechin gallate (EGCG) that induces PLAGL1 gene demethylation and promotes the apoptosis of pheochromocytoma (PCC) in PC12 cell line. Methods: The PC12 cells were treated with 25, 50, 75, 100, and 150 μg/mL EGCG for 48 hours. MSP was used to examine PLAGL1 gene methylation and an MTT assay was performed to detect the cell proliferation. The cell apoptosis was detected using flow cytometry. The mRNA and protein expressions of DNMT1, PLAGL1, Wnt, and β-catenin were detected using RT-quantitative PCR and Western blot. Results: EGCG dose-dependently reduced the cell viability and reversed PLAGL1 gene hypermethylation in PC12 cells (P<0.05). The cell apoptosis was significantly increased in PC12 cells treated with EGCG. The EGCG treatment restored the expressions of PLAGL1 and downregulated the expression of DNMT1, Wnt, and β-catenin in PC12 cells (P<0.05). Conclusion: The EGCG induces the demethylation process of PLAGL1 gene through down-regulating DNMT1 and restores the PLAGL1 mRNA and protein expression. The Wnt/β-catenin signaling pathway is involved in the regulation of PCC cell apoptosis promoted by EGCG inducing PLAGL1 gene demethylation.     

A Re-evaluation of the “Oncogenic” Nature of Wnt/β-catenin Signaling in Melanoma and Other Cancers

In cancer, Wnt/β-catenin signaling is ubiquitously referred to as an “oncogenic” pathway that promotes tumor progression. This review examines how the regulation and downstream effects of Wnt/β-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma. We emphasize that the cellular homeostasis of Wnt/β-catenin signaling may represent a more appropriate concept than the simplified view of the Wnt/β-catenin pathway as either oncogenic or tumor-suppressing. Ultimately, a more refined understanding of the contextual regulation of Wnt/β-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit.     

β-elemene Induces Caspase-dependent Apoptosis in Human Glioma Cells in vitro through the Upregulation of Bax and Fas/ FasL and Downregulation of Bcl-2

Background: β-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects invarious cancer cell lines. However, the activity of β-elemene against glioma cells remains unclear. In the presentstudy, we assessed effects of β-elemene on human glioma cells and explored the underlying mechanism. Materialsand Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay andcolony formation assay to detect the effect of β-elemene at different doses and times. Fluorescence microscopywas used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cyclingwere analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed toinvestigated the influence of β-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experimentwas divided into two groups: the blank control group and β-elemne treatment group. Results: With increase inthe concentration of β-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentrationof inhibited cell viability (IC50) was 48.5 μg/mL for 24h. β-elemene could induce cell cycle arrest in the G0/G1phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation ofcaspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, whilethe anti-apoptotic Bcl-2 was downregulated after treatment with β-elemene at both mRNA and protein levels.Furthermore, proliferation and colony formation by U87 cells were inhibited by β-elemene in a time and doesdependentmanner. Conclusions: Our results indicate that β-elemene inhibits growth and induces apoptosis ofhuman glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasLand Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptoticcascades.     

Opisthorchis viverrini Infection Activates the PI3K/ AKT/PTEN and Wnt/β-catenin Signaling Pathways in a Cholangiocarcinogenesis Model

Opisthorchis viverrini (Ov) infection is the major etiological factor for cholangiocarcinoma (CCA), especiallyin northeast Thailand. We have previously reported significant involvement of PI3K/AKT/PTEN and Wnt/β-catenin in human CCA tissues. The present study, therefore, examined the expression and activation of PI3K/AKT/PTEN and Wnt/β-catenin signaling components during Ov-induced cholangiocarcinogenesis in a hamsteranimal model. Hamsters were divided into two groups; non-treated and Ov plus NDMA treated. The resultsof immunohistochemical staining showed an upregulation of PI3K/AKT signaling as determined by elevatedexpression of the p85α-regulatory and p110α-catalytic subunits of PI3K as well as increased expression andactivation of AKT during cholangiocarcinogenesis. Interestingly, the staining intensity of activated AKT (p-AKT)increased in the apical regions of the bile ducts and strong staining was detected where the liver fluke resides.Moreover, PTEN, a negative regulator of PI3K/AKT, was suppressed by decreased expression and increasedphosphorylation during cholangiocarcinogenesis. We also detected upregulation of Wnt/β-catenin signaling asdetermined by increased positive staining of Wnt3, Wnt3a, Wnt5a, Wnt7b and β-catenin, corresponded withthe period of cholangiocarcinogenesis. Furthermore, nuclear staining of β-catenin was observed in CCA tissues.Our results suggest the liver fluke infection causes chronic inflammatory conditions which lead to upregulationof the PI3K/AKT and Wnt/β-catenin signaling pathways which may drive CCA carcinogenesis. These resultsprovide useful information for drug development, prevention and treatment of CCA.     

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.     

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of β-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.     

Advances in Chibby' s inhibition in the Wnt/β-catenin singaling pathway

It is well known that canonical Wnt/β-catenin signaling pathway plays a key role in tumori-genesis. Recent studies show that Chibby as an antagonist of the Wnt/β-catenin signaling pathway is a kind of nuclear protein that has been conserved throughout evolution. Chibby inhibits Wnt/β-catenin pathway at least through two distinct molecular pathways. Thus Chibby could inhibit the progression of tumors whose Wnt/β-catenin pathway is abnormally activated. This provids a new perspective in tumor research.     

Daidzein Induces Intrinsic Pathway of Apoptosis along with ER α/β Ratio Alteration and ROS Production

Background: Low risk of breast cancer is observed among females consuming a moderate quantity of soy throughout their life. The present study was conducted to evaluate the anticancer potential of Daidzein, one of the major Isoflavones in soy using Human breast cancer cells MCF-7. Methods: MCF-7 were subjected to various doses of Daidzein treatment to determine the IC50 value. Onset of apoptosis was ascertained by AnnexinV assay and caspase 3/7 activity post treatment. Expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 was also assessed to further confirm apoptotic mode of cell death. ROS production post treatment with Daidzein was assessed to ascertain the apoptosis via intrinsic pathway. Expression of ER α and ER β was evaluated by western blot analysis. Results: Human breast cancer cells MCF-7 were found to be sensitive to Daidzein treatment, with an IC50 value of 50µM. Increased percentage of treated cells stained with Annexin V confirmed apoptosis mediated cell death. Activity of Caspase 3/7 activity was found to be 1.4-fold higher in Daidzein treated cells than control cells, confirming apoptosis. Daidzein caused over expression of Bax and down-regulated expression of Bcl2. There has been an outburst of ROS in Daidzein treated cells indicating that Daidzein induces apoptosis via intrinsic pathway. A decrease in the expression of ER α and increase in levels of ER β has been observed which are conducive indicator of apoptosis. Conclusions: In conclusion, the present study suggests that Daidzein induces apoptosis in MCF-7 cells by mitochondrial pathway along with lowering the ratio of ER α/β and an outburst of Reactive Oxygen Species(ROS).     

Crosstalk between Wnt/β-catenin and Hedgehog/Gli signaling pathways in colon cancer and implications for therapy

Wnt/β-catenin and Hedgehog/Gli signalings play key roles in multiple biogenesis such as embryonic development and tissue homeostasis. Dysregulations of these 2 pathways are frequently found in most cancers, particularly in colon cancer. Their crosstalk has been increasingly appreciated as an important mechanism in regulating colon cancer progression. Our studies into the link between Wnt/β-catenin and Hedgehog/Gli signalings in colon cancer revealed several possible crosstalk points and suggested potential therapeutic strategies for colon cancer.     

The Wnt/β-catenin pathway regulates growth and maintenance of colonospheres

Background  

Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used.     

Blockage of Wnt/β-catenin Signaling Pathway in Colorectal Cancer Resistant Cells by Nitazoxanide Effects on Peptidylarginine Deiminases Expression

Background: Multidrug resistance (MDR) is a major cause of unsuccessful cancer treatment in which drugs are not effective. Therefore, it is necessary to identify the critical mechanisms of the development of MDR and target those with novel compounds. Accordingly, the current study is the first to investigate the combination effect and molecular mechanism of nitazoxanide (NTZ) and oxaliplatin (OXP) on LS174T/OXP-resistant cells. Methods: The effect of NTZ on OXP cytotoxicity in LS174T and LS174T/OXP cell lines was evaluated by MTT assay. Changes in expression levels of MDR1, MRP1, CTNNB1, peptidylarginine deiminase (PAD)2, and PAD4 genes and proteins were evaluated by RT-qPCR and western blotting methods, respectively. Lastly, the apoptosis assay was performed by flow cytometer. Results: OXP resistant and sensitive cells were identified based on the IC50 values (11567 nM vs. 1745 nM, p<0.05 for 24 h treatment; and 5161 nM vs. 882 nM, p<0.05 for 48 h incubation). The combination of NTZ and OXP for 48 h led to a reduction in IC50 values in resistant cells (2154 nM, p<0.05). The effect of NTZ plus OXP significantly decreased the expression of MDR1 (p<0.001), MRP1 (p<0.05), and CTNNB1 (p<0.001), while PAD2 and PAD4 expression was significantly increased (p<0.001). This combination therapy enhanced the percentage of the sub-G1 population (apoptosed) compared to other groups. Conclusion: The results showed that NTZ leads to notable upregulation of PAD2 and PAD4, which can disrupt the Wnt/β-catenin signaling pathway and reverse the MDR by reducing MDR1 and MRP1 expression.     

Wnt/Β-catenin and sex hormone signaling in endometrial homeostasis and cancer

A delicate balance between estrogen and progestagen signaling underlies proper functioning of the female reproductive tract and, in particular, the monthly re- and degenerative phases characteristic of the menstrual cycle. Here, we propose that the canonical Wnt/β-catenin signaling pathway may underlie this finely tuned hormonal equilibrium in endometrial homeostasis and, upon its constitutive activation, lead to neoplastic transformation of the endometrium. During the menstrual cycle, estradiol will enhance Wnt/β-catenin signaling in the proliferative phase, while progesterone inhibits Wnt/β-catenin signaling, thus restraining estrogens' proliferative actions, during the secretory phase. In case of enhanced or unopposed estrogen signaling, constitutive activation of Wnt/β-catenin signaling will trigger endometrial hyperplasia, which may develop further into endometrial cancer.     

Licochalcone A Induces Cholangiocarcinoma Cell Death Via Suppression of Nrf2 and NF-κB Signaling Pathways

Objective: To investigate the anti-tumor effect of licochalcone A (LCA) on proliferation and migration in cholangiocarcinoma (CCA) cells and to elucidate their underlying mechanisms. Methods: Human CCA cells, KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452 were used to study effect of LCA on proliferation and migration by a cytotoxicity assay, wound healing assay. Reactive oxygen species levels were evaluated using DHE-fluorescent probes. Proteins associated with cancer survival and progression were analyzed by immune blotting assay. Results: LCA suppressed proliferation and induced cell death in CCA cells including KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452. The CCAs cells were suppressed in association with LCA-induced accumulation of intracellular reactive oxygen species (ROS). Increased formation of ROS was causally related with suppression of Nrf2 and its down-stream antioxidant and cytoprotective enzymes. These effects may lead to the expression of Bax and release of cytochrome c and ensuring cell death.  Interestingly, LCA could also inhibit cell migration and cell cycle arrest at low concentrations. These effects were associated with down-regulation of NF-kB, STAT3 and their down-stream proteins, cyclin D1, VEGF, and ICAM-1. Conclusions: These results suggest that LCA has potential therapeutic activity in suppression of CCA cells.