Project description:The increased PD-L1 expression induces poorer prognosis in melanoma. The small molecule inhibitors of PD-1/PD-L1 pathways have been an encouraging drug development strategy because of good affinity and oral bioavailability without immunogenicity and immunotoxicities of PD-1/PD-L1 antibodies. In this study, we studied the effects of PCC0208025 (BMS202), a small molecule inhibitor of PD-L1, on PD-1/PD-L1 binding and the cytokines secretion in human CD3+ cells in vitro. We also investigated the antitumor and immunomodulatory activity of PCC0208025 and the pharmacokinetics properties in B16-F10 melanoma-bearing mice. The results showed that PCC0208025 inhibited the PD-1/PD-L1 proteins binding, and rescued PD-L1-mediated inhibition of IFN-γ production in human CD3+ T cells in vitro. Furthermore, in B16-F10 melanoma-bearing mice, PCC0208025 presented the antitumor effects, enhanced IFN-γ levels in plasma, increased the frequency of CD3+CD8+ T and CD8+IFN-γ+ T and the ratios of CD8+/Treg, and deceased the CD4+CD25+CD127low/- (Treg) number in tumor. Pharmacokinetics study found that PCC0208025 was absorbed and distributed into the tumors with much higher concentrations than those of the blockade against PD-1/PD-L1 binding. Our work suggests that PCC0208025 exhibited anti-tumor effects through inhibiting Treg expansion and increasing cytotoxic activity of tumor-infiltrating CD8+ T cells by the blockade of PD-1/PD-L1 binding, which may provide the pharmacological basis to develop small molecule inhibitors of PD-1/PD-L1 binding for PCC0208025 as a lead compound.
Project description:The chemokine CCL21 regulates immune and cancer cell migration through its receptor CCR7. The Ccl21a gene encodes the isoform CCL21-Ser, predominantly expressed in the thymic medulla and the secondary lymphoid tissues. This study examined the roles of CCL21-Ser in the antitumor immune response in Ccl21a-knockout (KO) mice. The Ccl21a-KO mice showed significantly decreased growth of B16-F10 and YUMM1.7 melanomas and increased growth of MC38 colon cancer, despite no significant difference in LLC lung cancer and EO771 breast cancer. The B16-F10 tumor in Ccl21a-KO mice showed melanoma-specific activated CD8+ T cell and NK cell infiltration and higher Treg counts than wild-type mice. B16-F10 tumors in Ccl21a-KO mice showed a reduction in the positive correlation between the ratio of regulatory T cells (Tregs) to activated CD8+ T cells and tumor weight. In Ccl21a-KO tumor, the intratumoral Tregs showed lower co-inhibitory receptors TIM-3 and TIGIT. Taken together, these results suggest that endogenous CCL21-Ser supports melanoma growth in vivo by maintaining Treg function and suppressing antitumor immunity by CD8+ T cells.
Project description:Cancer cells have acquired several pathways to escape from host immunity in the tumor microenvironment. Programmed death 1 (PD-1) receptor and its ligand PD-L1 are involved in the key pathway of tumor immune escape, and immune checkpoint therapy targeting PD-1 and PD-L1 has been approved for the treatment of patients with certain types of malignancies. Although PD-1 is a well-characterized receptor on T cells, the immune checkpoint receptor is also expressed on tumor-associated macrophages (TAM), a major immune component of the tumor microenvironment. In this study, we found significant diurnal oscillation in the number of PD-1-expressing TAMs collected from B16/BL6 melanoma-bearing mice. The levels of Pdcd1 mRNA, encoding PD-1, in TAMs also fluctuated in a diurnal manner. Luciferase reporter and bioluminescence imaging analyses revealed that a NF-κB response element in the upstream region of the Pdcd1 gene is responsible for its diurnal expression. A circadian regulatory component, DEC2, whose expression in TAMs exhibited diurnal oscillation, periodically suppressed NF-κB-induced transactivation of the Pdcd1 gene, resulting in diurnal expression of PD-1 in TAMs. Furthermore, the antitumor efficacy of BMS-1, a small molecule inhibitor of PD-1/PD-L1, was enhanced by administering it at the time of day when PD-1 expression increased on TAMs. These findings suggest that identification of the diurnal expression of PD-1 on TAMs is useful for selecting the most appropriate time of day to administer PD-1/PD-L1 inhibitors.ImplicationsSelecting the most appropriate dosing time of PD-1/PD-L1 inhibitors may aid in developing cancer immunotherapy with higher efficacy.
Project description:In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC)-modified sterically stabilized liposomes (SSLs) containing conjugated linoleic acid-paclitaxel (CLA-PTX). The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes' cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 μg/g) and 4 hours (1.17±0.28 μg/g) was 2.3- and 2.0-fold higher than that of CLA-PTX solution at 1 hour (0.79±0.06 μg/g) and 4 hours (0.58±0.04 μg/g). The value of the area under the curve for the first 24 hours in the tumors of iRGD-SSL-CLA-PTX-treated mice was significantly higher than that in the SSL-CLA-PTX and CLA-PTX solution-treated groups (P<0.01). The in vivo antitumor results indicated that iRGD-SSL-CLA-PTX significantly inhibited the growth of B16-F10 tumors compared with the SSL-CLA-PTX or CLA-PTX solution-treatment groups (P<0.01). The results of tumor-cell apoptosis showed that tumors from the iRGD-SSL-CLA-PTX-treated group exhibited more advanced cell apoptosis compared with the control, CLA-PTX solution-, and SSL-CLA-PTX-treated groups. In conclusion, the antitumor effect of iRGD-SSL-CLA-PTX was confirmed on B16-F10 melanoma in vitro and in vivo.
Project description:In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.
Project description:We developed and characterized a 3D collagen hydrogel model for B16.F10 melanoma tumors. Cells in this 3D environment exhibited lower proliferation than cells in the conventional 2D culture environment. Interestingly, the basal expression levels of several genes varied when compared to conventionally grown cells. In each growth environment, a significant number of melanoma cells were transfected by plasmid electroporation (electrotransfer), although expression could only be ascertained on the surface of the 3D constructs. Cellular responses to plasmid entry as demonstrated by pro-inflammatory cytokine and chemokine upregulation varied based on the growth environment, as did the mRNA levels of several putative DNA-specific pattern recognition receptors (DNA sensors). Unexpectedly, when plasmid DNA was delivered while cells where attached in the 2D or 3D environments, the mRNAs of the DNA sensor p204 and the inflammatory mediator TNFα were regulated in cells receiving pulses only. However, we were unable to confirm coordinate upregulation of TNFα and p204 proteins. This study confirms that cell responses differ significantly based on their environment, and demonstrates the difficulty of extending experimental observations between cell environments.
Project description:The hypoxia responsive protein BNIP3, plays an important role in promoting cell death and/or autophagy, ultimately resulting in a cancer type-dependent, tumour-enhancer or tumour-suppressor activity. We previously reported that in melanoma cells, BNIP3 regulates cellular morphology, mitochondrial clearance, cellular viability and maintains protein expression of CD47, a pro-cancerous, immunosuppressive 'don't eat me' signal. Surface exposed CD47 is often up-regulated by cancer cells to avoid clearance by phagocytes and to suppress immunogenic cell death (ICD) elicited by anticancer therapies. However, whether melanoma-associated BNIP3 modulates CD47-associated immunological effects or ICD has not been explored properly. To this end, we evaluated the impact of the genetic ablation of BNIP3 (i.e. BNIP3KD) in melanoma cells, on macrophage-based phagocytosis, polarization and chemotaxis. Additionally, we tested its effects on crucial determinants of chemotherapy-induced ICD (i.e. danger signals), as well as in vivo anticancer vaccination effect. Interestingly, loss of BNIP3 reduced the expression of CD47 both in normoxic and hypoxic conditions while macrophage phagocytosis and chemotaxis were accentuated only when BNIP3KD melanoma cells were exposed to hypoxia. Moreover, when exposed to the ICD inducer mitoxantrone, the loss of melanoma cell-associated BNIP3 did not alter apoptosis induction, but significantly prevented ATP secretion and reduced phagocytic clearance of dying cells. In line with this, prophylactic vaccination experiments showed that the loss of BNIP3 tends to increase the intrinsic resistance of B16-F10 melanoma cells to ICD-associated anticancer vaccination effect in vivo. Thus, normoxic vs. hypoxic and live vs. dying cell contexts influence the ultimate immunomodulatory roles of melanoma cell-associated BNIP3.
Project description:The patients diagnosed with melanoma have a bad prognosis for early regional invasion and distant metastases. Wogonin (5,7-dihydroxy-8-methoxyflavone) is one of the active components of flavonoids that extracts from Scutellariae radix. Several previous studies reported that wogonin possesses antitumor effect against leukemia, gastrointestinal cancer and breast cancer. In this study, we used melanoma cell B16-F10 to further investigate the anti-invasive and anti-migratory activity of wogonin. Our date showed that wogonin caused suppression of cell migration, adhesion, invasion and actin remodeling by inhibiting the expression of matrix metalloproteinase-2 and Rac1 in vitro. Wogonin also reduced the number of the tumor nodules on the whole surface of the lung in vivo. Furthermore, the examination of mechanism revealed that wogonin inhibited Extracellular Regulated protein Kinases and Protein Kinase B pathways, which are both medicated by Ras. Insulin-like growth factor-1-induced or tumor necrosis factor-α-induced invasion was also inhibited by wogonin. Therefore, the inhibitory mechanism of melanoma cell invasion by wogonin might be elucidated.
Project description:Roots of Glycyrrhiza uralensis have been used as herbal medicine and natural sweetener. By activity-guided phytochemical investigation of the extracts from G.uralensis root, ten flavonoids, namely GF-1⁻GF-10, of which five were prenylated flavonoids, were found to show antiproliferative effects in melanoma B16-F10 cells. Three of the prenylated flavonoids, namely GF-1, GF-4 and GF-9, significantly induced the differentiation of B16-F10 cells; the inductions included increase of tyrosinase activity, tyrosinase protein, and melanin content. In GF-1 and GF-9 induced melanoma differentiation, the phosphorylation of p38 MAPK (mitogen activated potein kinase) was identified; while GF-4 could trigger the phosphorylation of PI3K/AKT (phosphatidylinositol 3-kinase/Protein Kinase B) signaling. However, application of GF-6 to the melanoma cells did not induce differentiation; but which promoted cell apoptotic signaling, i.e., increase levels of cleaved-PRAP, cleaved-caspase 3, and cleaved-caspase 9. These results suggested that different types of prenylated flavonoids from G.uralensis might have potential anticancer effects against melanoma cells by acting through different signaling pathways.
Project description:PurposePrenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents.MethodsAP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells.ResultsAll the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination.ConclusionObtained results indicate that APs have a potential as non-viral vectors for cell transfection.