Corosolic acid inhibits cancer progression by decreasing the level of CDK19-mediated O-GlcNAcylation in liver cancer cells

Diabetes is an important risk factor for liver cancer, but its mechanism is unknown. Corosolic acid (CA) has been proven to have both hypoglycemic and antitumor effects, so revealing the function of CA can help us understand the relationship between diabetes and liver cancer. In previous studies, we confirmed that CA can effectively inhibit the expression of YAP, an important oncoprotein in HCC cells, and the proliferation of HCC cells. In addition, we also found that O-GlcNAcylation plays an indispensable role in HCC tumorigenesis. However, it is not clear whether CA can inhibit the effect of O-GlcNAcylation on HCC cells. In this study, the antitumor ability of CA was investigated by inhibiting the O-GlcNAcylation level and its corresponding mechanism. The results showed that HG (high glucose) could promote the proliferation of liver cancer cells, while CA could inhibit cell growth under HG conditions and tumor growth in a xenotransplantation model. CA can inhibit the activation of the HBP pathway and reduce the expression of YAP and OGT under HG conditions. Importantly, we found that CA can reduce YAP expression and O-GlcNAcylation by inhibiting the activity of CDK19. Overexpression of CDK19 partially reversed the CA-induced decrease in YAP and O-GlcNAcylation. This is the first evidence that CA can reduce the proliferative capacity of cells with high glucose levels and further inhibit tumor growth by inactivating the CDK19/YAP/O-GlcNAcylation pathway, suggesting that CA is a candidate drug for the development of treatments against diabetes-associated liver cancer.Subject terms: Oncogenes, Tumour angiogenesis     

Synthesis,characterization and cytotoxicity studies of 1,2,3-triazoles and 1,2,4-triazolo [1,5-a] pyrimidines in human breast cancer cells

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is essential for physiological functions of tissues and neovasculature. VEGFR signaling is associated with the progression of pathological angiogenesis in various types of malignancies, making it an attractive therapeutic target in cancer treatment. In the present work, we report the synthesis of 1,4-disubstituted 1,2,3-triazoles and 1,2,4-triazolo[1, 5-a]pyrimidine derivatives via copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and screened for their anticancer activity against MCF7 cells. We identified 1-(2′-ethoxy-4′-fluoro-[1,1′-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole (EFT) as lead cytotoxic agent against MCF7 cell lines with an IC₅₀ value of 1.69?µM. Further evaluation revealed that EFT induces cytotoxicity on Ishikawa, MDA-MB-231 and BT474 cells with IC₅₀ values of 1.97, 4.81 and 4.08?µM respectively. However, EFT did not induce cytotoxicity in normal lung epithelial (BEAS-2B) cells. Previous reports suggested that 1,2,3-triazoles are the inhibitors of VEGFR1 and therefore, we evaluated the effect of EFT on the expression of VEGFR1. The results demonstrated that EFT downregulates the expression of VEGFR1 in MCF7 cells. In summary, we identified a potent cytotoxic agent that imparts its antiproliferative activity by targeting VEGFR1 in breast cancer cells. The novel compound could serve as a lead structure in developing VEGFR1 inhibitors.     

Evaluation of anticancer effects of Juniperus communis extract on hepatocellular carcinoma cells in vitro and in vivo

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and accounts for the fourth leading cause of all cancer deaths. Scientific evidence has found that plant extracts seem to be a reliable choice due to their multitarget effects against HCC. Juniperus communis has been used for centuries in traditional medicine and its anticancer properties have been reported. As a result, the purpose of the study was to investigate the anticancer effect and mechanism of J. communis extract (JCo extract) on HCC in vitro and in vivo. In the present study, we found that JCo extract inhibited the growth of human HCC cells by inducing cell cycle arrest at the G₀/G₁ phase, extensive apoptosis and suppressing metastatic protein expressions in HCC cells. Moreover, the combinational treatment of JCo and VP-16 was found to enhance the anticancer effect, revealing that JCo extract might have the potential to be utilized as an adjuvant to promote HCC treatment. Furthermore, in vivo study, JCo extract significantly suppressed HCC tumor growth and extended the lifespan with no or low systemic and pathological toxicity. JCo extract significantly up-regulated the expression of pro-apoptotic proteins and tumor suppressor p53, suppressed VEGF/VEGFR autocrine signaling, down-regulated cell cycle regulatory proteins and MMP2/MMP9 proteins. Overall, our results provide a basis for exploiting JCo extract as a potential anticancer agent against HCC.     

Sophoridine suppresses lenvatinib-resistant hepatocellular carcinoma growth by inhibiting RAS/MEK/ERK axis via decreasing VEGFR2 expression

Hepatocellular carcinoma (HCC) is one of the most lethal cancer types with insufficient approved therapies, among which lenvatinib is a newly approved multi-targeted tyrosine kinase inhibitor for frontline advanced HCC treatment. However, resistance to lenvatinib has been reported in HCC treatment recently, which limits the clinical benefits of lenvatinib. This study aims to investigate the underlying mechanism of lenvatinib resistance and explore the potential drug to improve the treatment for lenvatinib-resistant (LR) HCC. Here, we developed two human LR HCC cell lines by culturing with long-term exposure to lenvatinib. Results showed that the vascular endothelial growth factor receptors (VEGFR)2 expression and its downstream RAS/MEK/ERK signalling were obviously up-regulated in LR HCC cells, whereas the expression of VEGFR1, VEGFR3, FGFR1-4 and PDGFRα/β showed no difference. Furthermore, ETS-1 was identified to be responsible for VEGFR2 mediated lenvatinib resistance. The cell models were further used to explore the potential strategies for restoration of sensitivity of lenvatinib. Sophoridine, an alkaloid extraction, inhibited the proliferation, colony formation, cell migration and increased apoptosis of LR HCC cells. In vivo and in vitro results showed Sophoridine could further sensitize the therapeutic of lenvatinib against LR HCC. Mechanism studies revealed that Sophoridine decreased ETS-1 expression to down-regulate VEGFR2 expression along with downstream RAS/MEK/ERK axis in LR HCC cells. Hence, our study revealed that up-regulated VEGFR2 expression could be a predicator of the resistance of lenvatinib treatment against HCC and provided a potential candidate to restore the sensitivity of lenvatinib for HCC treatment.     

Tryptanthrin Inhibits Angiogenesis by Targeting the VEGFR2-Mediated ERK1/2 Signalling Pathway

Angiogenesis is a key step for tumour growth and metastasis, and anti-angiogenesis has been proposed as an important strategy for cancer therapy. Tryptanthrin is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants and has been shown to possess anti-tumour activities on various cancer cell types. This study aims to investigate the in vitro and in vivo anti-angiogenic activities of tryptanthrin and to unravel its underlying molecular action mechanisms. Our results show that tryptanthrin inhibited the in vitro proliferation, migration, and tube formation of the human microvascular endothelial cells (HMEC-1) in a concentration-dependent manner and significantly suppressed angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that tryptanthrin reduced the expression of several pro-angiogenic factors (Ang-1, PDGFB and MMP2). Tryptanthrin was also found to suppress the VEGFR2-mediated ERK1/2 signalling pathway in HMEC-1 cells and molecular docking simulation indicated that tryptanthrin could bound to the ATP-binding site of VEGFR2. Collectively, the present study demonstrated that tryptanthrin exhibited both in vitro and in vivo anti-angiogenic activities by targeting the VEGFR2-mediated ERK1/2 signalling pathway and might have therapeutic potential for the treatment of angiogenesis-related diseases.     

RhoC/ROCK2 promotes vasculogenic mimicry formation primarily through ERK/MMPs in hepatocellular carcinoma

Vasculogenic mimicry (VM) results in the formation of an alternative circulatory system that can improve the blood supply to multiple malignant tumors, including hepatocellular carcinoma (HCC). However, the potential mechanisms of RhoC/ROCK in VM have not yet been investigated in HCC. Here, RhoC expression was upregulated in HCC tissues, especially the VM-positive (VM+) group, compared to noncancerous tissues (P < 0.01), and patients with high expression of RhoC had shorter survival times (P < 0.001). The knockdown of RhoC via short hairpin RNA (shRNA) in SK-Hep-1 cells significantly decreased VM formation and cell motility. In contrast, cell motility and VM formation were remarkably enhanced when RhoC was overexpressed in HepG2 cells. To further assess the potential role of ROCK1 and ROCK2 on VM, we stably knocked down ROCK1 or ROCK2 in MHCC97H cells. Compared to ROCK1 shRNA, ROCK2 shRNA could largely affect VM formation, cell motility and the key VM factors, as well as the epithelial-mesenchymal transition (EMT) markers in vitro and in vivo. Moreover, p-ERK, p-MEK, p-FAK, p-paxillin, MT1-MMP and MMP2 levels were clearly altered following the overexpression of RhoC, but ROCK2 shRNA had little effect on the expression of p-FAK, which indicated that RhoC regulates FAK/paxillin signaling, but not through ROCK2. In conclusion, our results show that RhoC/ROCK2 may have a major effect on VM in HCC via ERK/MMPs signaling and might be a potential therapeutic target for the treatment of HCC.     

GSTZ1 sensitizes hepatocellular carcinoma cells to sorafenib-induced ferroptosis via inhibition of NRF2/GPX4 axis

Increasing evidence supports that ferroptosis plays an important role in tumor growth inhibition. Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, has been shown to induce ferroptosis in hepatocellular carcinoma (HCC). However, some hepatoma cell lines are less sensitive to sorafenib-induced ferroptotic cell death. Glutathione S-transferase zeta 1 (GSTZ1), an enzyme in the catabolism of phenylalanine, suppresses the expression of the master regulator of cellular redox homeostasis nuclear factor erythroid 2-related factor 2 (NRF2). This study aimed to investigate the role and underlying molecular mechanisms of GSTZ1 in sorafenib-induced ferroptosis in HCC. GSTZ1 was significantly downregulated in sorafenib-resistant hepatoma cells. Mechanistically, GSTZ1 depletion enhanced the activation of the NRF2 pathway and increased the glutathione peroxidase 4 (GPX4) level, thereby suppressing sorafenib-induced ferroptosis. The combination of sorafenib and RSL3, a GPX4 inhibitor, significantly inhibited GSTZ1-deficient cell viability and promoted ferroptosis and increased ectopic iron and lipid peroxides. In vivo, the combination of sorafenib and RSL3 had a synergic therapeutic effect on HCC progression in Gstz1^−/− mice. In conclusion, this finding demonstrates that GSTZ1 enhanced sorafenib-induced ferroptosis by inhibiting the NRF2/GPX4 axis in HCC cells. Combination therapy of sorafenib and GPX4 inhibitor RSL3 may be a promising strategy in HCC treatment.Subject terms: Cancer therapeutic resistance, Cancer therapeutic resistance     

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the ‘wrapping’ pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.     

Vascular Endothelial Growth Factor-Receptor 1 Inhibition Aggravates Diabetic Nephropathy through eNOS Signaling Pathway in db/db Mice

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice.While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling.In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.     

Cyperenoic acid,a sesquiterpene derivative from Croton crassifolius,inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

BackgroundCyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated.PurposeTo investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis.Study designTargeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated.MethodsThe in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model.ResultsCyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2.ConlusionCyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.     

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p⁻) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p⁻ significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p⁻ can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p⁻ in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.     

Dual blockade of VEGFR1 and VEGFR2 by a novel peptide abrogates VEGF-driven angiogenesis,tumor growth,and metastasis through PI3K/AKT and MAPK/ERK1/2 pathway

Background

Neutralization of vascular endothelial growth factor receptor 1 (VEGFR1) and/or VEGFR2 is a widely used means of inhibiting tumor angiogenesis.

Phytotoxic cyanamide affects maize (Zea mays) root growth and root tip function: From structure to gene expression

Cyanamide (CA) is a phytotoxic compound produced by four Fabaceae species: hairy vetch, bird vetch, purple vetch and black locust. Its toxicity is due to complex activity that involves the modification of both cellular structures and physiological processes. To date, CA has been investigated mainly in dicot plants. The goal of this study was to investigate the effects of CA in the restriction of the root growth of maize (Zea mays), representing the monocot species. CA (3 mM) reduced the number of border cells in the root tips of maize seedlings and degraded their protoplasts. However, CA did not induce any significant changes in the organelle structure of other root cells, apart from increased vacuolization. CA toxicity was also demonstrated by its effect on cell cycle activity, endoreduplication intensity, and modifications of cyclins CycA2, CycD2, and histone HisH3 gene expression. In contrast, the arrangement of microtubules was not altered by CA. Treatment of maize seedlings with CA did not completely arrest mitotic activity, although the frequency of dividing cells was reduced. Furthermore, prolonged CA treatment increased the proportion of endopolyploid cells in the root tip. Cytological malformations were accompanied by an induction of oxidative stress in root cells, which manifested as enhanced accumulation of H₂O₂. Exposure of maize seedlings to CA resulted in an increased concentration of auxin and stimulated ethylene emission. Taken together, these findings suggested that the inhibition of root growth by CA may be a consequence of stress-induced morphogenic responses.     

Obatoclax,the pan-Bcl-2 inhibitor sensitizes hepatocellular carcinoma cells to promote the anti-tumor efficacy in combination with immune checkpoint blockade

Bcl-2 family proteins play critical roles in regulating lymphocyte development and maintain homeostasis, and have also been proved to be involved in various cancer types development. However, the role of Bcl-2 in hepatocellular carcinoma (HCC) development has not been clearly studied. Here, we reported the pan-Bcl-2 inhibitor, obatoclax could directly inhibit HCC growth in vitro. We further demonstrated in murine HCC model that obatoclax also suppressed HCC development in vivo. We also proved that although obatoclax inhibited T cells expansion, it had no influence on T cells activation in vivo. Mechanism study revealed that obatoclax sensitized HCC cells to T cell-mediated killing. Combination therapy of obatoclax with anti-PD-1 antibody synergistically suppressed HCC development and prolonged the survival rate of tumor-bearing mice. The combination therapy promoted T cells activation and effector cytokines expression both in spleen and tumor. In summary, our results proved that obatoclax sensitized HCC cells to T cell -mediated killing. Combination of obatoclax with immune checkpoint blockade served as a promising therapeutic strategy for HCC treatment.