Lycorine inhibits melanoma cell migration and metastasis mainly through reducing intracellular levels of β-catenin and matrix metallopeptidase 9

Metastatic melanoma accounts for 60% of death for skin cancer. Although great efforts have been made to treat the disease, effective drugs against metastatic melanoma still lack at the clinical setting. In the current study, we found that lycorine, a small molecule of isoquinoline alkaloid, significantly suppressed melanoma cell migration and invasion in vitro, and decreased the metastasis of melanoma cells to lung tissues in tumor-bearing mice, resulting in significant prolongation of the survival of the mice without obvious toxicity. Molecular mechanistic studies revealed that lycorine significantly reduced intracellular levels of β-catenin protein through degradation of the protein via the ubiquitin–proteasome pathway, and decreased the expression of β-catenin downstream prometastatic matrix metallopeptidase 9 and Axin2 genes. Collectively, our findings support the notion that targeting the oncogenic β-catenin by lycorine is a new option to inhibit melanoma cell metastasis, providing a good drug candidate potential for development novel therapeutics against metastatic melanoma.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-alpha and -beta, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

NRF2 as a determinant of cellular resistance in retinoic acid cytotoxicity

Clinical use of retinoic acids (RA) is hindered by toxicity possibly related to oxidative stress. Recently, RA at relatively low concentrations was shown to inhibit NRF2 and the expression of its target antioxidative genes. This raises the possibility that RA toxicity may result from cellular inability to cope with resultant oxidative stress. Using in vitro cell and in vivo mouse models, we report that RA, specifically all-trans-RA (atRA) at concentrations implicated in toxicity, can activate NRF2 and induce NRF2 target genes, particularly the subunits of the rate-limiting enzyme of glutathione biosynthesis, glutamate cysteine ligase (GCLM/GCLC). RNA interference-mediated silencing of NRF2, but not of retinoid X receptor-α and -β, reduced basal and atRA-induced GCLM/GCLC gene expression. Moreover, RA increased nuclear accumulation of NRF2, antioxidant response element (ARE) reporter activity, and NRF2 occupancy at AREs. 4-Hydroxynonenal, a lipid peroxidation product, was increased by RA. Inhibition of MEK1/ERK mitogen-activated protein kinases significantly suppressed atRA-induced NRF2 activation and ARE-regulated gene expression, reducing cell resistance against toxic concentrations of RA. NRF2-silenced cells were vulnerable to atRA-induced mitochondrial toxicity and apoptosis. In conclusion, toxic RA activates NRF2, thereby triggering an adaptive response against the resultant oxidative stress. NRF2 enhancement as a therapeutic target of retinoid toxicity awaits further investigation.     

Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, beta1-6 branching, and metastasis.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed enhanced metastatic potential in vivo, increased motility in vitro, increased ability to produce melanin, and responsiveness to melanocyte stimulating hormone compared with the parental Cloudman S91 melanoma cells. These hybrids also showed altered N-glycosylation consistent with a slower migration pattern of lysosome-associated membrane protein (LAMP-1) on electrophoretic gels. Because LAMP-1 is the major carrier of polylactosamine sugar structures, and synthesis of this complex sugar moiety indicates the extent of beta1,6 branch formation by beta1,6-N-acetyl-glucosaminyltransferase V (GnT-V), we analyzed the expression of GnT-V and beta1,6 branching in highly metastatic macrophage-fusion hybrids and compared with poorly metastatic ones. GnT-V was up-regulated in regard to both mRNA levels and enzymatic activity specifically in metastatic hybrids as well as parental macrophages compared with weakly metastatic hybrids and parental melanoma cells. Macrophages and metastatic hybrids also showed increased binding of the lectin L-phytohemagglutinin, which specifically binds to the beta1,6-branched sugar moiety. In addition, in metastatic hybrids there was increased cell surface expression of LAMP-1 and beta1 integrin, two prominent substrates for GnT-V also known to be associated with metastasis. Finally, exposure of metastatic hybrids in vitro to L-phytohemagglutinin or LAMP-1 completely eliminated melanocyte stimulating hormone/ isobutylmethyl xanthine-induced motility, suggesting a role for GnT-V in the motility of these cells. In summary, macrophage fusion with melanoma cells often increased metastatic potential, which was associated with enhanced expression of GnT-V and beta1,6-branching in glycoproteins. It is suggested that the known correlation with elevated GnT-V in both human and animal metastasis could, at least in some cases, reflect previous fusion of tumor cells with tumor-infiltrating macrophages, which, similar to malignant cells, show elevated expression of GnT-V and beta1,6-branched polylactosamines.     

LAZ3 protects cardiac remodeling in diabetic cardiomyopathy via regulating miR-21/PPARa signaling

Diabetes contributes to cardiovascular complications and the pathogenesis of cardiac remodeling that can lead to heart failure. We aimed to evaluate the functional role of LAZ3 in diabetic cardiomyopathy (DCM). Streptozotocin (STZ) was used to induce a diabetic mouse model. Three months after induction, the mice were subjected to retro-orbital venous plexus injection of adeno-associated virus 9 (AAV9) that overexpressed LAZ3. Six weeks after the infection, mouse hearts were removed to assess the degree of cardiac remodeling. LAZ3 was down-regulated in the diabetic mouse hearts and high glucose stimulated cardiomyocytes. Knock-down of LAZ3 in cardiomyocytes with LAZ3 siRNA reduced cell viability, increased the inflammatory response and induced oxidative stress and cell apoptosis. Overexpression of LAZ3 by infection with adeno-associated virus (AAV9)-LAZ3 protected against an inflammatory response, oxidative stress and cell apoptosis in both a high glucose stimulated in vitro study and diabetic mouse hearts. We found that LAZ3 increased the activation of PPARa, which increased PGC-1a activation and subsequently augmented NRF2 expression and nuclear translocation. This outcome was confirmed by NRF2 siRNA and a PPARa activator, since NRF2 siRNA abrogated the protective effects of LAZ3 overexpression, while the PPARa activator reversed the deteriorating phenotype of LAZ3 knock-down in both the in vitro and vivo study. Furthermore, LAZ3 decreased miR-21 expression, which resulted in PPARa activation, NRF2 expression and nuclear translocation. In conclusion, LAZ3 protects against cardiac remodeling in DCM by decreasing miR-21, thus regulating PPARa/NRF2 signaling.     

CD147 depletion down-regulates matrix metalloproteinase-11, vascular endothelial growth factor-A expression and the lymphatic metastasis potential of murine hepatocarcinoma Hca-F cells

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), which is a plasma membrane glycoprotein enriched on the surface of many malignant tumors promotes adhesion, invasion and metastasis of tumor cells. In addition, tumor-associated CD147 also induces vascular endothelial growth factors (VEGFs) expression. To investigate the possible role of CD147 in the mouse hepatocarcinoma cell line Hca-F with highly metastatic potential in the lymph nodes, we used an RNA interference (RNAi) approach to silence CD147 expression. The results showed that CD147 depletion in Hca-F cells resulted in the significantly decreased expression of matrix metalloproteinase-11 (MMP-11), VEGF-A at both mRNA and protein levels. The reduced CD147 expression also attenuated the invasive, adhesive, metastatic ability of Hca-F cells to lymph nodes both in vitro and in vivo. Our current findings reveal that the tumor biological marker CD147 functionally mediates MMP-11, VEGF-A expression and tumor lymphatic metastasis.     

Oxalomalate suppresses metastatic melanoma through IDH-targeted stress response to ROS

Melanoma is the most aggressive skin cancer due to a high propensity for metastasis, with a 10-year survival rate of less than 10%. The devastating clinical outcome and lack of effective preventative therapeutics for metastatic melanoma necessitate the development of new therapeutic strategies targeted to inhibit the regulatory circuits underlying the progression and metastasis of melanoma. Melanoma metastasis requires migration and invasion of the malignant tumour cells driven by proteolytic remodelling of the extracellular matrix (ECM) executed by matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9. Inhibiting components of these circuits defines new therapeutic opportunities for melanoma with metastatic malignancy. Oxalomalate (OMA) is a competitive inhibitor of NADP⁺-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signalling pathways regulated by reactive oxygen species (ROS). In this study, we investigated the therapeutic role of OMA in metastatic melanoma and the associated underlying mechanism of action. We report that OMA-mediated inhibition of IDH enzymes suppresses metastatic melanoma through inhibition of invasive cell migration based on MMP-9-mediated proteolytic remodelling of the ECM. In particular, our study provides the mechanistic foundation that OMA reduces the expression and secretion of MMP-9 through LKB1-mediated PEA3 degradation via the ROS-dependent ATM-Chk2-p53 signalling axis, resulting from inhibition of IDH enzymes. These results provide evidence that OMA targeting of the stress response to ROS by IDH inhibition is a promising therapy for the treatment of metastatic melanoma.     

MHY884, a newly synthesized tyrosinase inhibitor,suppresses UVB-induced activation of NF-κB signaling pathway through the downregulation of oxidative stress

The skin is the primary target of prolonged and repeated ultraviolet (UVB) irradiation which induces cutaneous inflammation and pigmentation. Nuclear factor κB (NF-κB) is the major factor mediating UVB-induced inflammatory responses through the expression of various proinflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We have previously reported that the synthetic novel compound 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) strongly suppressed tyrosinase activity and melanin synthesis in B16F10 melanoma cells. In the present study, we investigated the effect of MHY884 on the inhibition of UVB-induced NF-κB activation and its proinflammatory downstream proteins through the suppression of oxidative stress in an in vivo model of photoaging. Generation of reactive oxygen species (ROS) and peroxynitrite was measured in vitro and in B16F10 melanoma cells to verify the scavenging activity of MHY884. MHY884 suppressed oxidative stress both in vitro and in the melanoma cells in a dose-dependent manner. Next, melanin-possessing hairless mice were pre-treated with MHY884 and then irradiated with UVB repeatedly. Topical application of MHY884 attenuated UVB-induced oxidative stress, resulting in reduced NF-κB activity. Pre-treatment with MHY884 inhibited Akt and IκB kinase α/β signaling pathways, leading to decreased translocation and phosphorylation of p65, a subunit of NF-κB. This result correlated with the expression levels of iNOS and COX-2 in the skin of MHY884-treated mice. Thus, the novel tyrosinase inhibitor MHY884 suppressed NF-κB activation signaling pathway by scavenging UVB-induced oxidative stress. The discovery of MHY884, a novel tyrosinase inhibitor that targets NF-κB signaling, is significant, because this compound is a promising protective agent against UVB-induced skin damage.     

d-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis

D: -Penicillamine (3,3-dimethyl-D: -cysteine; DP) is an FDA-approved redox-active D: -cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L: -cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2α, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.     

Effects of raf kinase inhibitor protein expression on metastasis and progression of human epithelial ovarian cancer

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis, we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently, the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result, the cell proliferation, anchorage-independent growth, cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore, the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation, migration, or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA.     

Silencing of Diphthamide Synthesis 3 (Dph3) Reduces Metastasis of Murine Melanoma

Melanoma is the most dangerous skin cancer due to its highly metastatic potential and resistance to chemotherapy. Currently, there is no effective treatment for melanoma once it is progressed to metastatic stage. Therefore, further study to elucidate the molecular mechanism underlying the metastasis of melanoma cells is urgently required for the improvement of melanoma treatment. In the present study, we found that diphthamide synthesis 3 (Dph3) is involved in the metastasis of B16F10 murine melanoma cells by insertional mutagenesis. We demonstrated that Dph3 disruption impairs the migration of B16F10 murine melanoma cells. The requirement of Dph3 in the migration of melanoma cells was further confirmed by gene silencing with siRNA in vitro. In corresponding to this result, overexpression of Dph3 significantly promoted the migratory ability of B16F10 and B16F0 melanoma cells. Moreover, down regulation of Dph3 expression in B16F10 melanoma cells strikingly inhibits their cellular invasion and metastasis in vivo. Finally, we found that Dph3 promotes melanoma migration and invasion through the AKT signaling pathway. To conclude, our findings suggest a novel mechanism underlying the metastasis of melanoma cells which might serve as a new intervention target for the treatment of melanoma.