Absent in melanoma 2 suppresses epithelial-mesenchymal transition via Akt and inflammasome pathways in human colorectal cancer cells

Absent in melanoma 2 (AIM2) is a critical component in natural immunity system and is closely related to cancer initiation and development. It has been shown that AIM2 inhibited colorectal cancer (CRC) development and cell proliferation. It remains unresolved how AIM2 acts on CRC metastasis. In this study, we assessed migration, invasion ability, and epithelial-mesenchymal transition (EMT) program upon AIM2 overexpression or knockdown in human CRC cells. Transwell assay demonstrated that upregulation of AIM2 reduced cell migration and invasion. Epithelial marker E-cadherin was augmented and mesenchymal markers vimentin, as well as Snail, were examined decreased by Western blot, real-time polymerase chain reaction, and immunofluorescence. Correspondingly, knockdown of AIM2 led to a reverse consequence. In addition, AIM2 regulated Akt phosphorylation and effects of AIM2 on cell invasion and EMT were recovered after administration of Akt inhibitor, suggesting that AIM2 suppressed EMT dependent on Akt pathway. In addition, caspase-1 inhibitor exposure indicated that AIM2 abrogated EMT through the inflammasome pathway as well. In summary, AIM2 suppressed EMT via Akt and inflammasome pathways in human CRC cells.     

Absent in melanoma 2-mediating M1 macrophages facilitate tumor rejection in renal carcinoma

Absent in melanoma 2 (AIM2) as an immune regulator for the regulation of tumor-associated macrophages (TAMs) function is unclear in tumor development. Here, the AIM2 function was investigated in TAMs-mediated malignant behaviors of renal carcinoma. The correlation analysis result showed that the AIM2 expression in TAMs was negatively correlated with the percentages of M2-like polarization phenotype in human or murine renal cancer specimens. By the cocultured assay with bone marrow-derived macrophages (BMDMs) and Renca cells, overexpression of AIM2 in macrophages enhanced the inflammasome activation and reversed the phenotype from M2 to M1. Compared with BMDMs-Ctrl cocultured group, BMDMs-AIM2 cocultured group showed reduced tumor cell proliferation and migration. The blockade of inflammasome activation by the inhibitor Ac-YVAD-CMK abrogated AIM2-mediated M1 polarization and the inhibition of tumor cell growth. To evaluate the therapeutic efficacy of AIM2-mediated M1 macrophages in vivo, BMDMs-AIM2 were intravenously injected into subcutaneous Renca-tumor mice. The results showed that the infiltration of M1 TAMs was increased and tumor growth was suppressed in BMDMs-AIM2-treated mice when compared with BMDMs-Ctrl-treat mice. Accordingly, the blockade of inflammasome activation reduced the anti-tumor activities of BMDMs-AIM2. Moreover, the lung metastases of renal carcinoma were suppressed by the administration of BMDMs-AIM2 accompanied with the reduced tumor foci. These results demonstrated that AIM2 enhanced TAMs polarization switch from anti-inflammatory M2 phenotypy to pro-inflammatory M1 through inflammasome signaling activation, thus exerting therapeutic intervention in renal carcinoma models. Our results provide a possible molecular mechanism for the modulation of TAMs polarization in tumor microenvironment and open a new potential therapeutic approach for renal cancer.     

Curcumin enhances the anti‐cancer effects of Paris Saponin II in lung cancer cells

Objectives

To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer.

Materials and Methods

The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells.

Results

The combination displayed a synergistic anti‐cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF‐α to induce cancer cells apoptosis, and up‐regulated IGFBP‐1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI‐H446 cells. Meanwhile, the combination suppressed PCNA and NF‐κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI‐H460 and NCI‐H446 cells, enhanced the phosphorylation of JNK in NCI‐H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI‐H520 cells.

Conclusions

PSII combined with CUR had a synergistic anti‐cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

     

NF‐κB/NKILA signaling modulates the anti‐cancerous effects of EZH2 inhibition

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.     

Suppression of epidermal growth factor receptor‐mediated β‐catenin nuclear accumulation enhances the anti‐tumor activity of phosphoinositide 3‐kinase inhibitor in breast cancer

Phosphoinositide 3‐kinase (PI3K) signaling is frequently deregulated in breast cancer and plays a critical role in tumor progression. However, resistance to PI3K inhibitors in breast cancer has emerged, which is due to the enhanced β‐catenin nuclear accumulation. Until now, the mechanisms underlying PI3K inhibition‐induced β‐catenin nuclear accumulation remains largely unknown. In the present study, we found inhibition of PI3K with LY294002 promoted β‐catenin nuclear accumulation in MCF‐7 and MDA‐MB‐231 breast cancer cells. Combining PI3K inhibitor LY294002 with XAV‐939, an inhibitor against β‐catenin nuclear accumulation, produced an additive anti‐proliferation effect against breast cancer cells. Subsequent experiments suggested β‐catenin nuclear accumulation induced by PI3K inhibition depended on the feedback activation of epidermal growth factor receptor (EGFR) signaling pathway in breast cancer cells. Inhibition of EGFR phosphorylation with Gefitinib enhanced anti‐proliferation effect of PI3K inhibitor LY294002 in MCF‐7 and MDA‐MB‐231 cells. Taken together, our findings may elucidate a possible mechanism explaining the poor outcome of PI3K inhibitors in breast cancer treatment.     

Progress in the research of p53 tumour suppressor activity controlled by Numb in triple‐negative breast cancer

Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.     

2‐methoxyestradiol‐mediated anti‐tumor effect increases osteoprotegrin expression in osteosarcoma cells

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2‐Methoxyestradiol (2‐ME), a naturally occurring metabolite of 17β‐estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2‐ME actions, we studied the effect of 2‐ME treatment on OPG gene expression in human osteosarcoma cells. 2‐ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2‐ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3‐, 1.9‐, 2.8‐, and 2.5‐fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2‐ME treatment. The effect of 2‐ME on osteosarcoma cells was ligand‐specific as parent estrogen, 17β‐estradiol and a tumorigenic estrogen metabolite, 16α‐hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co‐treating osteosarcoma cells with OPG protein did not further enhance 2‐ME‐mediated anti‐tumor effects. OPG‐released in 2‐ME‐treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2‐ME‐mediated anti‐proliferative effects in osteosarcoma cells, but rather participates in anti‐resorptive functions of 2‐ME in bone tumor environment. J. Cell. Biochem. 109: 950–956, 2010. © 2010 Wiley‐Liss, Inc.     

Dioscin elicits anti‐tumour immunity by inhibiting macrophage M2 polarization via JNK and STAT3 pathways in lung cancer

Tumour‐associated macrophage (TAM) is an important component in tumour microenvironment. Generally, TAM exhibits the function of M2‐like macrophage, which was closely related to angiogenesis and tumour progression. Dioscin, a natural steroidal saponin, has shown its powerful anti‐tumour activity recently. However, the mechanism of dioscin involved in immune regulation is still obscure. Here, we observed dioscin induced macrophage M2‐to‐M1 phenotype transition in vitro and inhibited IL‐10 secretion. Meanwhile, the phagocytosis of macrophages was enhanced. In subcutaneous lung tumour models, dioscin inhibited the augmentation of M2 macrophage populations. Furthermore, dioscin down‐regulated STAT3 and JNK signalling pathways in macrophages in vitro. In BMDMs, activating JNK and inhibiting STAT3 induce macrophages to M1 polarization while inhibiting JNK and activating STAT3 to M2 polarization. Additionally, condition mediums from dioscin‐pre‐treated macrophages inhibited the migration of 3LL cells and the tube‐formation capacity of HUVECs. What's more, dioscin‐mediated macrophage polarization inhibited the in vivo metastasis of 3LL cells. In conclusion, dioscin may act as a new anti‐tumour agent by inhibiting TAMs via JNK and STAT3 pathways in lung cancer.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

JS‐K induces reactive oxygen species‐dependent anti‐cancer effects by targeting mitochondria respiratory chain complexes in gastric cancer

As a nitric oxide (NO) donor prodrug, JS‐K inhibits cancer cell proliferation, induces the differentiation of human leukaemia cells, and triggers apoptotic cell death in various cancer models. However, the anti‐cancer effect of JS‐K in gastric cancer has not been reported. In this study, we found that JS‐K inhibited the proliferation of gastric cancer cells in vitro and in vivo and triggered mitochondrial apoptosis. Moreover, JS‐K induced a significant accumulation of reactive oxygen species (ROS), and the clearance of ROS by antioxidant reagents reversed JS‐K‐induced toxicity in gastric cancer cells and subcutaneous xenografts. Although JS‐K triggered significant NO release, NO scavenging had no effect on JS‐K‐induced toxicity in vivo and in vitro. Therefore, ROS, but not NO, mediated the anti‐cancer effects of JS‐K in gastric cancer. We also explored the potential mechanism of JS‐K‐induced ROS accumulation and found that JS‐K significantly down‐regulated the core proteins of mitochondria respiratory chain (MRC) complex I and IV, resulting in the reduction of MRC complex I and IV activity and the subsequent ROS production. Moreover, JS‐K inhibited the expression of antioxidant enzymes, including copper‐zinc‐containing superoxide dismutase (SOD1) and catalase, which contributed to the decrease of antioxidant enzymes activity and the subsequent inhibition of ROS clearance. Therefore, JS‐K may target MRC complex I and IV and antioxidant enzymes to exert ROS‐dependent anti‐cancer function, leading to the potential usage of JS‐K in the prevention and treatment of gastric cancer.     

Synergy between anti‐CCL2 and docetaxel as determined by DW‐MRI in a metastatic bone cancer model

Metastatic prostate cancer continues to be the second leading cause of cancer death in American men with an estimated 28,660 deaths in 2008. Recently, monocyte chemoattractant protein‐1 (MCP‐1, CCL2) has been identified as an important factor in the regulation of prostate metastasis. CCL2, shown to attract macrophages to the tumor site, has a direct promotional effect on tumor cell proliferation, migration, and survival. Previous studies have shown that anti‐CCL2 antibodies given in combination with docetaxel were able to induce tumor regression in a pre‐clinical prostate cancer model. A limitation for evaluating new treatments for metastatic prostate cancer to bone is the inability of imaging to objectively assess response to treatment. Diffusion‐weighted MRI (DW‐MRI) assesses response to anticancer therapies by quantifying the random (i.e., Brownian) motion of water molecules within the tumor mass, thus identifying cells undergoing apoptosis. We sought to measure the treatment response of prostate cancer in an osseous site to docetaxel, an anti‐CCL2 agent, and combination treatments using DW‐MRI. Measurements of tumor apparent diffusion coefficient (ADC) values were accomplished over time during a 14‐day treatment period and compared to response as measured by bioluminescence imaging and survival studies. The diffusion data provided early predictive evidence of the most effective therapy, with survival data results correlating with the DW‐MRI findings. DW‐MRI is under active investigation in the pre‐clinical and clinical settings to provide a sensitive and quantifiable means for early assessment of cancer treatment outcome. J. Cell. Biochem. 107: 58–64, 2009. © 2009 Wiley‐Liss, Inc.     

Down‐regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L^?1, 50 μL), the combination with metformin (60 mmol L^?1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.     

Novel functions for 2‐phenylbenzimidazole‐5‐sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis

In this study, we investigated the effects and molecular mechanisms of 2‐phenylbenzimidazole‐5‐sulphonic acid (PBSA), an ultraviolet B protecting agent used in sunscreen lotions and moisturizers, on ovarian cancer cell responses and tumour angiogenesis. PBSA treatment markedly blocked mitogen‐induced invasion through down‐regulation of matrix metalloproteinase (MMP) expression and activity in ovarian cancer SKOV‐3 cells. In addition, PBSA inhibited mitogen‐induced cell proliferation by suppression of cyclin‐dependent kinases (Cdks), but not cyclins, leading to pRb hypophosphorylation and G₁ phase cell cycle arrest. These anti‐cancer activities of PBSA in ovarian cancer cell invasion and proliferation were mediated by the inhibition of mitogen‐activated protein kinase kinase 3/6‐p38 mitogen‐activated protein kinase (MKK3/6‐p38^MAPK) activity and subsequent down‐regulation of MMP‐2, MMP‐9, Cdk4, Cdk2 and integrin β1, as evidenced by treatment with p38^MAPK inhibitor SB203580. Furthermore, PBSA suppressed the expression and secretion of vascular endothelial growth factor in SKOV‐3 cells, leading to inhibition of capillary‐like tubular structures in vitro and angiogenic sprouting ex vivo. Taken together, our results demonstrate the pharmacological effects and molecular targets of PBSA on modulating ovarian cancer cell responses and tumour angiogenesis, and suggest further evaluation and development of PBSA as a promising chemotherapeutic agent for the treatment of ovarian cancer.