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Chrysin is a natural flavonoid with anti-cancer effects. Despite its beneficial effects, little information is available regarding its immunogenic cell death (ICD) properties. In this work, we hypothesized that chrysin can potentiate radiotherapy(RT)-induced immunogenicity in melanoma cell line (B16-F10). We examined the effects of chrysin alone and in combination with radiation on ICD induction in B16-F10 cells. Cell viability was assessed using an MTT assay. Cell apoptosis and calreticulin (CRT) exposure were determined using flow cytometry. Western blotting and ELISA assay were employed to examine changes in protein expression. Combination therapy exhibited a synergistic effect, with an optimum combination index of 0.66. The synergistic anti-cancer effect correlated with increased cell apoptosis in cancer cells. Compared to the untreated control, chrysin alone and in combination with RT induced higher levels of DAMPs, such as CRT, HSP70, HMGB1, and ATP. The protein expression of p-STAT3/STAT3 and PD-L1 was reduced in B16-F10 cells exposed to chrysin alone and in combination with RT. Conditioned media from B16-F10 cells exposed to mono-and combination treatments elicited IL-12 secretion in dendritic cells (DCs), inducing a Th1 response. Our findings revealed that chrysin could induce ICD and intensify the RT-induced immunogenicity., (© 2024. The Author(s).)

Penghu cactus (Opuntia dillenii [Ker.] Haw) is a cactus plant that commonly grows in Penghu Island, Taiwan, Republic of China (ROC). However, still lack of scientific study on the Opuntia dillenii [Ker.] Haw extract on skin-whitening-associated tyrosinase activity and melanin production. The activities of its extract in melanogenesis were investigated in this article. In this experiment, we used an extract from the Penghu cactus (Opuntia dillenii [Ker.] Haw) to study its tyrosinase inhibition, anti-melanin generation, UV-protection effects and wound healing capacity in B16-F10 melanocytes. Without reducing cell growth greatly or causing cell death, 20 g/L cactus extract effectively inhibited the melanin production of B16-F10 cells, and melanogenesis was induced by 3-isobutyl-1-methylxanthine. The cactus extract could also promote cell proliferation. Cactus extract treatment decreased the mRNA expression of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) and increased that of transforming growth factor β (TGF-β). Thus, it could reduce cell melanin production and promote cell growth but by also reducing IGF-1 and VEGF mRNA expression, may reduce wound scarring and prevent tumor proliferation and swelling. Increasing TGF-β mRNA expression can help increase collagen to remove wrinkles and help in wound healing. Skin patch test results agreed with in vitro results with B16-F10 melanoma cells. The cactus extract significantly inhibited tyrosinase activity and reduced melanin production, showing a whitening effect on skin tests. Cactus may be a good natural candidate for inhibiting melanin production and promoting cell proliferation., (© 2023. The Author(s).)

A double disulfide tethering depsipeptide dimer, romipeptide A (1) was prepared by NaOH catalyzed dimerization of romidepsin. Its structure was determined by analysis of NMR and HR-ESI-MS data as well as single crystal X-ray diffraction. Bioassay results showed that 1 exhibited good cytotoxic activity against two tumor cell lines B16 and HCT116. This study reported the single crystal data of 1 for the first time. The facile preparation of 1 afforded enough amount for its further biological evaluations., (© 2021. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities., (© 2021. The Author(s).)

Low-dose metronomic chemotherapy (LDMC) inhibits tumor angiogenesis and growth by targeting tumor-associated endothelial cells, but the molecular mechanism has not been fully elucidated. Here, we examined the functional role of regulated in development and DNA damage responses 1 (REDD1), an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in LDMC-mediated endothelial cell dysfunction. Low-dose doxorubicin (DOX) treatment induced REDD1 expression in cultured vascular and lymphatic endothelial cells and subsequently repressed the mRNA expression of mTORC1-dependent translation of vascular endothelial growth factor receptor (Vegfr)-2/3, resulting in the inhibition of VEGF-mediated angiogenesis and lymphangiogenesis. These regulatory effects of DOX-induced REDD1 expression were additionally confirmed by loss- and gain-of-function studies. Furthermore, LDMC with DOX significantly suppressed tumor angiogenesis, lymphangiogenesis, vascular permeability, growth, and metastasis in B16 melanoma-bearing wild-type but not Redd1-deficient mice. Altogether, our findings indicate that REDD1 is a crucial determinant of LDMC-mediated functional dysregulation of tumor vascular and lymphatic endothelial cells by translational repression of Vegfr-2/3 transcripts, supporting the potential therapeutic properties of REDD1 in highly progressive or metastatic tumors., (© 2021. The Author(s).)

Brucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer., (© 2021. The Author(s).)

Despite the widespread use of the blockade of immune checkpoints, for a significant number of cancer patients, these therapies have proven ineffective, presumably due to the immunosuppressive nature of the tumor microenvironment (TME). Critical drivers of immune escape in the TME include tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which not only mediate immune suppression, but also facilitate metastatic dissemination and impart resistance to immunotherapies. Thus, strategies that convert them into tumor fighters may offer great therapeutic potential. In this study, we evaluated whether pharmacologic modulation of macrophage phenotype by HDAC inhibitors (HDACi) could produce an anti-tumor effect. We demonstrated that low-dose HDACi trichostatin-A (TSA) markedly reshaped the tumor immune microenvironment by modulating the suppressive activity of infiltrating macrophages and inhibiting the recruitment of MDSCs in various tumors. These actions, in turn, augmented anti-tumor immune responses and further enhanced anti-tumor effects of immunotherapies. HDAC inhibition, however, also upregulated PD-L1, thereby limiting the beneficial therapeutic effects. Indeed, combining low-dose TSA with anti-PD-L1 in this model significantly enhanced the durability of tumor reduction and prolonged survival of tumor-bearing mice, compared with the effect of either treatment alone. These data introduce HDAC inhibition as a potential means to harness the anti-tumor potential of macrophages in cancer therapy.

Novel biocompatible and brush copolymers have been developed for cancer treatment using its controlled drug-release potential. Polyurethane graft on linear dextrin has been synthesized to control the hydrophilic-hydrophobic balance for regulated drug delivery. The properties of the graft copolymers have been tuned through graft density. The prepared grafts are thermally stable and mechanically strong. An injectable hydrogel has been developed by embedding the drug-loaded brush copolymers in methyl cellulose to better control the release for a prolonged period, importantly by keeping the drug release at a constant rate. Cellular studies indicate the biocompatible nature of the brush copolymers whose controlled and slow release of drug exhibit significant cytotoxic effects on cancer cells. Endocytosis of drug tagged contrast agent indicates greater transport of biologically active material inside cell as observed through cellular uptake studies. In vivo studies on melanoma mice exhibit the real efficacy of the controlled drug release from the injectable hydrogel with significant melanoma suppression without any side effects as opposed to severe toxic effects observed in conventional chemotherapy. Special application method of drug-loaded hydrogel just beneath the tumor makes this system incredibly effective through confinement. Thus, brush copolymer injectable hydrogel is a promising vehicle for control release of drug for cancer treatment in future.

Metastases account for the majority of cancer deaths. Bone represents one of the most common sites of distant metastases, and spinal bone metastasis is the most common source of neurological morbidity in cancer patients. During metastatic seeding of cancer cells, endothelial-tumor cell interactions govern extravasation to the bone and potentially represent one of the first points of action for antimetastatic treatment. The ephrin-B2-EphB4 pathway controls cellular interactions by inducing repulsive or adhesive properties, depending on forward or reverse signaling. Here, we report that in an in vivo metastatic melanoma model, ephrin-B2-mediated activation of EphB4 induces tumor cell repulsion from bone endothelium, translating in reduced spinal bone metastatic loci and improved neurological function. Selective ephrin-B2 depletion in endothelial cells or EphB4 inhibition increases bone metastasis and shortens the time window to hind-limb locomotion deficit from spinal cord compression. EphB4 overexpression in melanoma cells ameliorates the metastatic phenotype and improves neurological outcome. Timely harvesting of bone tissue after tumor cell injection and intravital bone microscopy revealed less tumor cells attached to ephrin-B2-positive endothelial cells. These results suggest that ephrin-B2-EphB4 communication influences bone metastasis formation by altering melanoma cell repulsion/adhesion to bone endothelial cells, and represents a molecular target for therapeutic intervention.

Angiogenesis inhibitors, such as the receptor tyrosine kinase (RTK) inhibitor sunitinib, target vascular endothelial growth factor (VEGF) signaling in cancers. However, only a fraction of patients respond, and most ultimately develop resistance to current angiogenesis inhibitor therapies. Activity of alternative pro-angiogenic growth factors, acting via RTK or G-protein coupled receptors (GPCR), may mediate VEGF inhibitor resistance. The phosphoinositide 3-kinase (PI3K)β isoform is uniquely coupled to both RTK and GPCRs. We investigated the role of endothelial cell (EC) PI3Kβ in tumor angiogenesis. Pro-angiogenic GPCR ligands were expressed by patient-derived renal cell carcinomas (PD-RCC), and selective inactivation of PI3Kβ reduced PD-RCC-stimulated EC spheroid sprouting. EC-specific PI3Kβ knockout (ΕC-βKO) in mice potentiated the sunitinib-induced reduction in subcutaneous growth of LLC1 and B16F10, and lung metastasis of B16F10 tumors. Compared to single-agent sunitinib treatment, tumors in sunitinib-treated ΕC-βKO mice showed a marked decrease in microvessel density, and reduced new vessel formation. The fraction of perfused mature tumor microvessels was increased in ΕC-βKO mice suggesting immature microvessels were most sensitive to combined sunitinib and PI3Kβ inactivation. Taken together, EC PI3Kβ inactivation with sunitinib inhibition reduces microvessel turnover and decreases heterogeneity of the tumor microenvironment, hence PI3Kβ inhibition may be a useful adjuvant antiangiogenesis therapy with sunitinib.

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

Mitochondrial dysfunction and significant changes in metabolic pathways accompany cancer development and are responsible for maintaining the tumor microenvironment. Normal mitochondria can trigger intrinsic apoptosis by releasing cytochrome c into the cytosol. The survival of malignant cells highly depends on the suppression of this function. We validated that A250, a highly purified fraction of fermented wheat germ extract (FWGE), increases the carbon flux into the mitochondria, the expression of key elements of the Krebs cycle and oxidative phosphorylation (OXPHOS). The increased respiratory chain activity is related to the mitochondria's ability to release cytochrome c into the cytosol, which triggers the apoptotic cascade. The 68% tumor growth inhibitory effect observed in the murine melanoma study is related to this effect, as proteomic analysis validated similar changes in mitochondrial protein levels in the isolated tumor tissue samples. Blood count data indicated that this effect was not accompanied by general toxicity. This study is significant, as it shows that a highly concentrated form of FWGE is an effective agent that increases normal mitochondrial functionality. The lack of hepatotoxic and general toxic effects makes A250 an excellent candidate targeting mitochondria function in cancer therapy.

Despite advances in therapy for melanoma, heterogeneous responses with limited durability represent a major gap in treatment outcomes. The purpose of this study was to determine whether alteration in tumor blood flow could augment drug delivery and improve antitumor responses in a regional model of melanoma. This approach to altering tumor blood flow was termed "dynamic control." Dynamic control of tumor vessels in C57BL/6 mice bearing B16 melanoma was performed using volume expansion (saline bolus) followed by phenylephrine. Intravital microscopy (IVM) was used to observe changes directly in real time. Our approach restored blood flow in non-functional tumor vessels. It also resulted in increased chemotherapy (melphalan) activity, as measured by formation of DNA adducts. The combination of dynamic control and melphalan resulted in superior outcomes compared to melphalan alone (median time to event 40.0 vs 25.0 days, respectively, p = 0.041). Moreover, 25% (3/12) of the mice treated with the combination approach showed complete tumor response. Importantly, dynamic control plus melphalan did not result in increased adverse events. In summary, we showed that dynamic control was feasible, directly observable, and augmented antitumor responses in a regional model of melanoma. Early clinical trials to determine the translational feasibility of dynamic control are ongoing.

Systemic toxicity caused by conventional chemotherapy is often regarded as one of the major challenges in the treatment of cancer. Over years, the trigger-based modality has gained much attention as it holds the spatiotemporal control over release and internalization of the drug. In this article, we are reporting an increase in the anti-tumor efficacy of curcumin due to ultrasound pulses. MDA MB 231 breast cancer and B16F10 melanoma cells were incubated with lecithin-based curcumin encapsulated nanoemulsions and exposed to ultrasound in the presence and absence of microbubble. Ultrasound induced sonoporation enhanced the cytotoxicity of curcumin in MDA MB 231 and B16F10 cancer cells in the presence of microbubble by 100- and 64-fold, respectively. To study the spatiotemporal delivery of curcumin, we developed B16F10 melanoma subcutaneous tumor on both the flanks of C57BL/6 mice but only the right tumor was exposed to ultrasound. Insonation of the right tumor spatially enhanced the cytotoxicity and enabled the substantial regression of the right tumor compared to the unexposed left tumor which grew continuously in size. This study showed that the ultrasound has the potential to target and increase the drug's throughput to the tumor and enable effective treatment.

We explore a novel strategy of activating immune signaling through increased micronuclei formation utilizing a cell cycle checkpoint inhibitor to drive cell cycle progression following ionizing radiation. The Chk1/2 inhibitor AZD7762 is used to abrogate radiation therapy (RT)-induced G2/M cell cycle arrest in multiple cell lines and, we find that this therapeutic combination promotes increased micronuclei formation in vitro and subsequently drives increased type I interferon signaling and cytotoxic T-cell activation. In vivo studies using B16-F10 melanoma cancer cells implanted in C57/BL6 mice demonstrate improved rates of tumor control at the abscopal (unirradiated) site, located outside of the radiation field, only in the AZD7762 + RT group, with a corresponding reduction in mean tumor volume, increase in the CD8 T-cell population, and immune activated gene signaling. Our results demonstrate that targeted inhibition of cell cycle checkpoint activation following ionizing radiation drives increased production of immunogenic micronuclei, leading to systemic tumor response with potential future clinical benefit.

Anti-vascular endothelial growth factor (VEGF) therapy shows antitumor activity against various types of solid cancers. Several resistance mechanisms against anti-VEGF therapy have been elucidated; however, little is known about the mechanisms by which the acquired resistance arises. Here, we developed new anti-VEGF therapy-resistant models driven by chronic expression of the mouse VEGFR2 extracellular domain fused with the human IgG4 fragment crystallizable (Fc) region (VEGFR2-Fc). In the VEGFR2-Fc-expressing resistant tumors, we demonstrated that the FGFR2 signaling pathway was activated, and pericytes expressing high levels of FGF2 were co-localized with endothelial cells. Lenvatinib, a multiple tyrosine kinase inhibitor including VEGFR and FGFR inhibition, showed marked antitumor activity against VEGFR2-Fc-expressing resistant tumors accompanied with a decrease in the area of tumor vessels and suppression of phospho-FGFR2 in tumors. Our findings reveal the key role that intercellular FGF2 signaling between pericytes and endothelial cells plays in maintaining the tumor vasculature in anti-VEGF therapy-resistant tumors.

Modulating the tumor microenvironment to promote an effective immune response is critical in managing any type of tumor. Melanoma is an aggressive skin cancer and the incidence rate is increasing worldwide. Potent protease inhibitors have recently been extensively researched as potential therapeutic agents against various cancers. EgKI-1 is a potent Kunitz type protease inhibitor identified from the canine tapeworm Echinococcus granulosus that has shown anti-cancer activities in vivo. In this study we show that EgKI-1 significantly reduced the growth of melanoma in the B16-F0 mouse model and was not toxic to normal surrounding tissue. Moreover, EgKI-1 treatment significantly reduced survivin expression levels and increased the CD8+ T cell population in draining axillary lymph nodes. Therefore, EgKI-1 potentially reduces tumor growth by inducing apoptosis and modulating the tumor microenvironment, and has potential for development as an intra-lesional treatment for melanoma.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Attenuated Listeria monocytogenes (Lm at -LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lm at -LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lm at -LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lm at -LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8 + T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3'-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH 2 -Gly(ArgLeu) 4 -TCAA displaying ribonuclease activity to its 5'-end. Designed miRNases efficiently cleaved miRNA targets, exhibiting Pyr-X specificity, and cleavage specificity had strong dependence on the miRNA sequence in the site of peptide location. In vitro, designed miRNases do not prevent cleavage of miRNA bound with the conjugate by RNase H, and more than an 11-fold enhancement of miRNA cleavage by the conjugate is observed in the presence of RNase H. In murine melanoma cells, miRNase silences mmu-miR-17 with very high efficiency as a result of miR-17 cleavage by miRNase and by recruited RNase H. Thus, miRNases provide a system of double attack of the miRNA molecules, significantly increasing the efficiency of miRNA downregulation in the cells in comparison with antisense oligonucleotide.

Shift metabolism profile from mitochondrial oxidative phosphorylation to aerobic glycolysis (Warburg effect) is a key for tumor cell growth and metastasis. Therefore, suppressing the tumor aerobic glycolysis shows a great promise in anti-tumor therapy. In the present study, we study the role of shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, in inhibiting tumor aerobic glycolysis and thus tumor growth. We found that shikonin dose-dependently inhibited glucose uptake and lactate production in Lewis lung carcinoma (LLC) and B16 melanoma cells, confirming the inhibitory effect of shikonin on tumor aerobic glycolysis. Treatment of shikonin also decreased tumor cell ATP production. Furthermore, pyruvate kinase M2 (PKM2) inhibitor or activator respectively altered the effect of shikonin on tumor cell aerobic glycolysis, suggesting that suppression of cell aerobic glycolysis by shikonin is through decreasing PKM2 activity. Western blot analysis confirmed that shikonin treatment reduced tumor cell PKM2 phosphorylation though did not reduce total cellular PKM2 level. In vitro assay also showed that shikonin treatment significantly promoted tumor cell apoptosis compared to untreated control cells. Finally, when mice implanted with B16 cells were administered with shikonin or control vehicle, only shikonin treatment significantly decreased B16 tumor cell growth. In conclusion, this study demonstrates that shikonin inhibits tumor growth in mice by suppressing PKM2-mediated aerobic glycolysis.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation 2,3 . Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U 34 ) tRNA (U 34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U 34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U 34 enzymes. Mechanistically, U 34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U 34 enzymes and HIF1α. Together, these results demonstrate that U 34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

In this study a novel method was implemented and investigated in order to destroy cancer cells inside the mouse body on a clinical level. In the case of in-vitro study, MTT assay was employed to discover an effective dose of applied plasma and distinguish the plasma effect in direct and in indirect treatments. Tumor growth was also measured in in-vivo section so that the effectiveness of direct and indirect treatments could be compared. Furthermore, an investigation was conducted to study the interferences between a conventional method (chemotherapy) and plasma treatment so as to increase the effectiveness of treatment inside the body. Hematoxylin and Eosin, Flow Cytometry, TUNEL and Western Blot assay were used to investigate any cell alteration and the impact of various treatment methods on cancer cell and amount of their apoptosis and protein levels. Radiology and CT scan images were taken to determine the final tumor volume. The results showed a significant cell death and substantial reduction in tumor growth in direct plasma treatment in comparison with indirect plasma treatment. Eventually, dramatic destruction of cancer cells was observed while using of indirect plasma-chemotherapy combination, thus introducing an effective method for deep tissue tumors can be introduced.

Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumour microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumour responses to radiotherapy. A poorly understood feature of these responses is the delayed onset (days), in contrast to the acute DNA-damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate-limiting steps that are essential for DNA-damage-induced inflammation. Here we show that cell cycle progression through mitosis following double-stranded DNA breaks leads to the formation of micronuclei, which precede activation of inflammatory signalling and are a repository for the pattern-recognition receptor cyclic GMP-AMP synthase (cGAS). Inhibiting progression through mitosis or loss of pattern recognition by stimulator of interferon genes (STING)-cGAS impaired interferon signalling. Moreover, STING loss prevented the regression of abscopal tumours in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

Peptide-drug discovery using host-defense peptides becomes promising against antibiotic-resistant pathogens and cancer cells. Here, we customized the therapeutic activity of bovine cathelicidin-5 targeting to bacteria, protozoa, and tumor cells. The membrane dependent conformational adaptability and plasticity of cathelicidin-5 is revealed by biophysical analysis and atomistic simulations over 200 μs in thymocytes, leukemia, and E. coli cell-membranes. Our understanding of energy-dependent cathelicidin-5 intrusion in heterogeneous membranes aided in designing novel loss/gain-of-function analogues. In vitro findings identified leucine-zipper to phenylalanine substitution in cathelicidin-5 (1-18) significantly enhance the antimicrobial and anticancer activity with trivial hemolytic activity. Targeted mutants of cathelicidin-5 at kink region and N-terminal truncation revealed loss-of-function. We ensured the existence of a bimodal mechanism of peptide action (membranolytic and non-membranolytic) in vitro. The melanoma mouse model in vivo study further supports the in vitro findings. This is the first structural report on cathelicidin-5 and our findings revealed potent therapeutic application of designed cathelicidin-5 analogues.

Autophagy-induced cancer cell death has become a novel strategy for the development of cancer therapeutic drugs. Numerous studies have indicated that green tea polyphenols induce both autophagy and apoptosis in a variety of cancer cells. Here, we synthesized a series of green tea polyphenol analogues, among which JP8 was shown to potently activate autophagy. JP8 treatment had a stronger effect on apoptosis in B16-F10 melanoma cells than that in normal AML-12 hepatocytes. JP8 selectively resulted in reactive oxygen species (ROS) accumulation in B16-F10 cells, and this effect was associated with corresponding increases in key components of the ER stress-mediated apoptosis pathway. Pharmacological inhibition of ROS by N-acetyl-L-cysteine (NAC) attenuated JP8-induced autophagy and apoptosis, indicating an upstream role of ROS in JP8-induced autophagy. An in vivo study showed that JP8 had significant antitumor effects in a B16-F10 xenograft mouse model. Our results indicate that JP8 is a novel anticancer candidate with both autophagy and ROS induction activities., Competing Interests: The authors declare no competing financial interests.

Metabolomics is a comprehensive assessment of endogenous metabolites of a biological system in a holistic context. In this study, we evaluated the in vivo anti-melanoma activity of aqueous extract of Forsythiae Fructus (FAE) and globally explored the serum metabolome characteristics of B16-F10 melanoma-bearing mice. UPLC/Q-TOF MS combined with pattern recognition approaches were employed to examine the comprehensive metabolic signatures and differentiating metabolites. The results demonstrated that FAE exhibited remarkable antitumor activity against B16-F10 melanoma in C57BL/6 mice and restored the disturbed metabolic profile by tumor insult. We identified 17 metabolites which were correlated with the antitumor effect of FAE. Most of these metabolites are involved in glycerophospholipid metabolisms. Notably, several lysophosphatidylcholines (LysoPCs) significantly decreased in tumor model group, while FAE treatment restored the changes of these phospholipids to about normal condition. Moreover, we found that lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX) were highly expressed in melanoma, and FAE markedly down-regulated their expression. These findings indicated that modulation of glycerophospholipid metabolisms may play a pivotal role in the growth of melanoma and the antitumor activity of FAE. Besides, our results suggested that serum LysoPCs could be potential biomarkers for the diagnosis and prognosis of melanoma and other malignant tumors.

Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.

The development of reactive oxygen species (ROS)-responsive drug delivery and drug release has gradually attracted much attention in recent years as a promising therapeutic strategy. Singlet oxygen ((1)O2) as the major ROS species is widely used in photodynamic therapy (PDT) of cancer. In the present study, we introduce a combined treatment using ROS-sensitive thioketal (TK) linkage as a linker between upconversion nanoparticles (UNs)-based PDT and doxorubicin (DOX)-based chemotherapy. UNs can not only play a role in PDT, but can also be used as a nanocarrier for drug delivery of DOX. Moreover, the products of (1)O2 during PDT are able to cleave TK linker inducing the release of DOX which can further achieve the goal of chemotherapy. By using this (1)O2-responsive nanocarrier delivery system, DOX can easily reach the tumor site and be accumulated in the nuclei to effectively kill the cancer cells, and therefore decreasing the side effects of chemotherapy on the body. Thus, PDT also has the function of controlling drug release in this combination treatment strategy. Compared with monotherapy, the combination of PDT with chemotherapy also possesses excellent drug loading capability and anticancer efficiency.

Bacteria-based living anticancer agents have emerged as promising therapeutics. However, the functional role of autophagy in bacterial cancer therapy has been little investigated. In this study, Salmonella VNP20009 induced autophagy in B16F10 cells, which is an unfavorable factor in bacterial cancer therapy. Inhibiting the induction of autophagy by chloroquine or siRNA in bacterial cancer therapy dose- and time-dependently promoted cell death. The combined therapy of VNP20009 and chloroquine not only enhanced the bacterial tumor targeting ability but also facilitated the infiltration of immune cells into the tumor. Our results showed that the combined therapy of VNP20009 and chloroquine could significantly inhibit tumor growth and prolong mouse survival time. This study provides a novel strategy for improving the anti-cancer efficacy of bacterial cancer therapy.

Climacostol, a compound produced by the ciliated protozoan Climacostomum virens, displayed cytotoxic properties in vitro. This study demonstrates that it has anti-tumour potential. Climacostol caused a reduction of viability/proliferation of B16-F10 mouse melanoma cells, a rapidly occurring DNA damage, and induced the intrinsic apoptotic pathway characterised by the dissipation of the mitochondrial membrane potential, the translocation of Bax to the mitochondria, the release of Cytochrome c from the mitochondria, and the activation of Caspase 9-dependent cleavage of Caspase 3. The apoptotic mechanism of climacostol was found to rely on the up-regulation of p53 and its targets Noxa and Puma. In vivo analysis of B16-F10 allografts revealed a persistent inhibition of tumour growth rate when melanomas were treated with intra-tumoural injections of climacostol. In addition, it significantly improved the survival of transplanted mice, decreased tumour weight, induced a remarkable reduction of viable cells inside the tumour, activated apoptosis and up-regulated the p53 signalling network. Importantly, climacostol toxicity was more selective against tumour than non-tumour cells. The anti-tumour properties of climacostol and the molecular events associated with its action indicate that it is a powerful agent that may be considered for the design of pro-apoptotic drugs for melanoma therapy.

The poor efficacy of the in vivo anti-tumor immune response has been partially attributed to ineffective T-cell responses mounted against the tumor. Fas-FasL-dependent activation-induced cell death (AICD) of T cells is believed to be a major contributor to compromised anti-tumor immunity. The molecular mechanisms of AICD are well-investigated, yet the possibility of regulating AICD for cancer therapy remains to be explored. In this study, we show that histone deacetylase inhibitors (HDACIs) can inhibit apoptosis of CD4(+) T cells within the tumor, thereby enhancing anti-tumor immune responses and suppressing melanoma growth. This inhibitory effect is specific for AICD through suppressing NFAT1-regulated FasL expression on activated CD4(+) T cells. In gld/gld mice with mutation in FasL, the beneficial effect of HDACIs on AICD of infiltrating CD4(+) T cells is not seen, confirming the critical role of FasL regulation in the anti-tumor effect of HDACIs. Importantly, we found that the co-administration of HDACIs and anti-CTLA4 could further enhance the infiltration of CD4(+) T cells and achieve a synergistic therapeutic effect on tumor. Therefore, our study demonstrates that the modulation of AICD of tumor-infiltrating CD4(+) T cells using HDACIs can enhance anti-tumor immune responses, uncovering a novel mechanism underlying the anti-tumor effect of HDACIs.

Concerns related to pulmonary toxicity and non-specificity of nanoparticles have limited their clinical applications for aerosol delivery of chemotherapeutics in lung cancer. We hypothesized that pulmonary surfactant mimetic nanoparticles that offer pH responsive release specifically in tumor may be a possible solution to overcome these issues. We therefore developed lung surfactant mimetic and pH responsive lipid nanovesicles for aerosol delivery of paclitaxel in metastatic lung cancer. 100-200 nm sized nanovesicles showed improved fusogenicity and cytosolic drug release, specifically with cancer cells, thereby resulting in improved cytotoxicity of paclitaxel in B16F10 murine melanoma cells and cytocompatibility with normal lung fibroblasts (MRC 5). The nanovesicles showed airway patency similar to that of endogenous pulmonary surfactant and did not elicit inflammatory response in alveolar macrophages. Their aerosol administration while significantly improving the biodistribution of paclitaxel in comparison to Taxol (i.v.), also showed significantly higher metastastes inhibition (~75%) in comparison to that of i.v. Taxol and i.v. Abraxane. No signs of interstitial pulmonary fiborisis, chronic inflammation and any other pulmonary toxicity were observed with nanovesicle formulation. Overall, these nanovesicles may be a potential platform to efficiently deliver hydrophobic drugs as aerosol in metastatic lung cancer and other lung diseases, without causing pulmonary toxicity.

Eupatorium fortunei has long been used to treat nausea and poor appetite, and has been prescribed as a diuretic and detoxifying drug in Chinese medicine. Recent studies have demonstrated that E. fortunei possesses anti-bacterial, anti-oxidant, and anti-diabetic activities, as well as cytotoxicity to human leukemia cells. However, at non-toxic concentrations, the effects of an aqueous extract of E. fortunei (WEF) on the metastatic and angiogenic potential of malignant tumor cells have not been reported. In this study, we found that WEF suppressed the metastatic properties, including anchorage-independent colony formation, migration, and invasion, by downregulating the proteolytic activity of MMP-9. NF-κB activation and the phosphorylation of p38 and JNK were reduced significantly by WEF. Additionally, WEF inhibited tumor-induced angiogenesis markedly, affecting HUVEC migration, tube formation by HUVECs, and microvessel sprouting from rat aortic rings via a reduction in VEGF in tumors. In a pulmonary metastasis model, daily administration of WEF at 50 mg/kg markedly decreased metastatic colonies of intravenously injected B16F10 cells on the lung surface in C57BL/6J mice. Further, none of the WEF-administered mice exhibited systemic toxicity. Taken together, our results indicate that WEF is a potential therapeutic herbal product that may be useful for controlling malignant metastatic cancer.

SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING protein required for the activity of Cullin-RING ligase (CRL). Our recent study showed that Sag total knockout caused embryonic lethality at E11.5-12.5 days with associated defects in vasculogenesis. Whether Sag is required for de novo vasculogenesis in embryos and angiogenesis in tumors is totally unknown. Here, we report that Sag endothelial deletion also causes embryonic lethality at E15.5 with poor vasculogenesis. Sag deletion in primary endothelial cells (ECs) or knockdown in MS-1 ECs inhibits migration, proliferation and tube formation, with p27 accumulation being responsible for the suppression of migration and proliferation. Furthermore, Sag deletion significantly inhibits angiogenesis in an in vivo Matrigel plug assay, and tumor angiogenesis and tumorigenesis in a B16F10 melanoma model. Finally, MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that inhibits CRL, suppresses in vitro migration, proliferation and tube formation, as well as in vivo angiogenesis and tumorigenesis. Taken together, our study, using both genetic and pharmaceutical approaches, demonstrates that Sag is essential for embryonic vasculogenesis and tumor angiogenesis, and provides the proof-of-concept evidence that targeting Sag E3 ubiquitin ligase may have clinical value for anti-angiogenesis therapy of human cancer.

Aim: To investigate the anticancer effects of S115, a novel heteroaromatic thiosemicarbazone compound in vitro and in vivo., Methods: The anti-proliferative action of S115 was analyzed in 12 human and mouse cancer cell lines using MTT assay. Autograft and xenograft cancer models were made by subcutaneous inoculation of cancer cells into mice or nude mice. The mice were orally treated with S115 (2, 8, 32 mg·kg(-1)·d(-1)) for 7 d, and the tumor size was measured every 3 d. Cell apoptosis and cell cycle distribution were examined using flow cytometry, gene expression profile analyses, Western blots and RT-PCR., Results: The IC50 values of S115 against 12 human and mouse cancer cell lines ranged from 0.3 to 6.6 μmol/L. The tumor growth inhibition rate caused by oral administration of S115 (32 mg·kg(-1)·d(-1)) were 89.7%, 81.7%, 78.4% and 77.8%, respectively, in mouse model of B16 melanoma, mouse model of Colon26 colon cancer, nude mouse model of A549 lung cancer and nude mouse model of SK-OV-3 ovarian cancer. Furthermore, oral administration of S115 (7.5 mg·kg(-1)·d(-1)) synergistically enhanced the anticancer effects of cyclophosphamide, cisplatin, or 5-fluorouracil in mouse model of S180 sarcoma. Treatment of A549 human lung cancer cells with S115 (1.5 μmol/L) induced G0/G1 cell cycle arrest, and increased apoptosis. Furthermore, S115 downregulated the level of ubiquitin, and upregulated the level of Tob2 in A549 cells., Conclusion: S115 exerts anticancer effects against a variety of cancer cells in vitro and in grafted cancer models by inducing apoptosis, downregulating ubiquitin and upregulating Tob2.

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Opioids are effective analgesics for the management of moderate to severe cancer pain. Here we show that κ opioid receptor (KOR) agonists act as anti-angiogenic factors in tumors. Treatment with KOR agonists, U50,488H and TRK820, significantly inhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation by suppressing VEGFR2 expression. In contrast, treatment with a μ opioid receptor agonist, DAMGO, or a δ opioid receptor agonist, SNC80, did not prevent angiogenesis in HUVECs. Lewis lung carcinoma (LLC) or B16 melanoma grafted in KOR knockout mice showed increased proliferation and remarkably enhanced tumor angiogenesis compared with those in wild type mice. On the other hand, repeated intraperitoneal injection of TRK820 (0.1-10 μg/kg, b.i.d.) significantly inhibited tumor growth by suppressing tumor angiogenesis. These findings indicate that KOR agonists play an important role in tumor angiogenesis and this knowledge could lead to a novel strategy for cancer therapy.

Aim: To investigate whether luteolin, a highly prevalent flavonoid, reverses the effects of epithelial-mesenchymal transition (EMT) in vitro and in vivo and to determine the mechanisms underlying this reversal., Methods: Murine malignant melanoma B16F10 cells were exposed to 1% O(2) for 24 h. Cellular mobility and adhesion were assessed using Boyden chamber transwell assay and cell adhesion assay, respectively. EMT-related proteins, such as E-cadherin and N-cadherin, were examined using Western blotting. Female C57BL/6 mice (6 to 8 weeks old) were injected with B16F10 cells (1×10(6) cells in 0.2 mL per mouse) via the lateral tail vein. The mice were treated with luteolin (10 or 20 mg/kg, ip) daily for 23 d. On the 23rd day after tumor injection, the mice were sacrificed, and the lungs were collected, and metastatic foci in the lung surfaces were photographed. Tissue sections were analyzed with immunohistochemistry and HE staining., Results: Hypoxia changed the morphology of B16F10 cells in vitro from the cobblestone-like to mesenchymal-like strips, which was accompanied by increased cellular adhesion and invasion. Luteolin (5-50 μmol/L) suppressed the hypoxia-induced changes in the cells in a dose-dependent manner. Hypoxia significantly decreased the expression of E-cadherin while increased the expression of N-cadherin in the cells (indicating the occurrence of EMT-like transformation), which was reversed by luteolin (5 μmol/L). In B16F10 cells, luteolin up-regulated E-cadherin at least partly via inhibiting the β3 integrin/FAK signal pathway. In experimental metastasis model mice, treatment with luteolin (10 or 20 mg/kg) reduced metastatic colonization in the lungs by 50%. Furthermore, the treatment increased the expression of E-cadherin while reduced the expression of vimentin and β3 integrin in the tumor tissues., Conclusion: Luteolin inhibits the hypoxia-induced EMT in malignant melanoma cells both in vitro and in vivo via the regulation of β3 integrin, suggesting that luteolin may be applied as a potential anticancer chemopreventative and chemotherapeutic agent.

Aim: DNAs containing unmethylated CpG motifs can stimulate innate and adaptive immunity. The aim of this study was to investigate the immunostimulatory and anti-neoplasm effects of a novel CpG oligodeoxynucleotide, ODN10, in tumor-bearing mice., Methods: B16 melanoma-bearing C57BL/6 mice were administered ip or sc with ODN10 or conventional CpG ODN1826 on the indicated days post inoculation. The animal survival rate and the inhibitory effect on tumor growth were observed in vivo. B and T lymphocyte proliferation, natural killing cell cytotoxicity and the phagocytic ability of peritoneal macrophages from the animals were determined using [(3)H]-thymidine incorporation assay, 4-h (51)Cr release assay and neutral red chromometry method, respectively. The serum levels of IL-12, IL-4 and IgE were quantified using ELISA assays. Histological examination of tumor tissues was performed after HE staining, and the expression of PCNA, CD63, and CD80 in tumor tissues was analyzed with immunohistochemistry., Results: ODN10 (1, 5 and 25 mg/kg) significantly inhibited the growth and metastasis of the tumor, and significantly prolonged the survival of tumor-bearing mice, as compared with ODN1826. The immune status was suppressed in tumor-bearing mice. Both ODN10 and ODN1826 significantly reversed the suppressed immunoactivities in tumor-bearing mice, which included promoting B and T lymphocyte proliferation, enhancing NK cell and peritoneal macrophage activities, inducing IL-12 secretion and inhibiting IL-4 and IgE secretion. Further, CpG ODNs decreased PCNA and CD63 expression while induced expression of CD80. ODN10 presented more potent activity, and displayed the most prominent immunostimulatory potential., Conclusion: ODN10 produces prominent immunomodulatory effects on cellular immunity in tumor-bearing mice, which might help reverse the established Th2-type responses to the Th1-type responses, thus may be used as a potent anti-tumor immunotherapy agent or adjuvant.

Dacarbazine (DTIC) is one of the most popular alkylating agents used for the treatment of malignant melanoma. DTIC induces apoptosis of melanoma cells via double-strand breaks (DSBs). Melanoma cells, however, tend to increase their expression of DNA repair molecules in order to be resistant to DTIC. Here, we show that DTIC increases expression of Rad51, but not Ku70, in a cultured B16-F10 mouse melanoma cell line in dose- and time-dependent manners. On introducing Rad51 short interfering RNA (siRNA) with the hemagglutinating virus of Japan envelope (HVJ-E) to B16-F10 cells, DSBs induced by DTIC treatment were not efficiently repaired and resulted in enhanced apoptotic cell death. Colony formation of B16-F10 cells that received Rad51 siRNA was significantly decreased by DTIC treatment as compared with cells that received scramble siRNA. In melanoma-bearing mice, the combination of three intratumoral injections of HVJ-E containing Rad51 siRNA and five intraperitoneal injections of DTIC at a clinical dose synergistically suppressed the tumors. Moreover, HVJ-E demonstrated anti-tumor immunity by inducing cytotoxic T lymphocytes to B16-F10 cells on administration of DTIC. These results suggest that the combination of chemotherapy with HVJ-E containing therapeutic molecules will provide a promising therapeutic strategy for patients bearing malignant tumors resistant to chemotherapeutic agents.

The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1α induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1α. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1α protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1α. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.

Melanoma cells depend on sustained proteasomal function for survival. However, bortezomib, the first proteasome inhibitor in clinical use, is not sufficient to improve the poor prognosis of metastatic melanoma patients. Since the proteasome is also expressed in all normal cell compartments, it is unclear how to enhance the efficacy of bortezomib without exacerbating secondary toxicities. Here, we present pharmacological and genetic analyses of mechanisms of resistance to proteasome inhibition. We focused on Bcl-2, Bcl-x(L) and Mcl-1 as main antiapoptotic factors associated with melanoma progression. Despite an efficient blockage of the proteasome, bortezomib could not counteract the intrinsically high levels of Bcl-2 and Bcl-x(L) in melanoma cells. Moreover, Mcl-1 was only downregulated at late time points after treatment. Based on these results, a combination treatment including (-)-gossypol, an inhibitor of Mcl-1/Bcl-2/Bcl-x(L), was designed and proven effective in vivo. Using a specific RNA interference approach, the survival of bortezomib-treated melanoma cells was found to rely primarily on Mcl-1, and to a lesser extent on Bcl-x(L) (but not on Bcl-2). Importantly, neither Mcl-1 nor Bcl-x(L) inactivation affected the viability of normal melanocytes. This hierarchical requirement of Bcl-2 family members for the maintenance of normal and malignant cells offers a therapeutic window to overcome melanoma chemoresistance in a tumor cell-selective manner.

Owing to its impressive ability to kill tumor cells, especially in combination with interferon-gamma (IFNgamma), tumor necrosis factor (TNF) is widely appreciated as being a potential systemic therapeutic for the treatment of cancer. On the other hand, owing to its proinflammatory activities, administration of TNF leads to many systemic side effects and eventually to a potentially lethal systemic inflammatory response syndrome (SIRS). However, systemic treatment of tumor-bearing mice with TNF/IFNgamma in combination with BB-94 (a broad-spectrum metalloproteinase inhibitor) confers protection against TNF/IFNgamma-induced mortality, whereas preserving the antitumor activity. In this study, we investigated the effect of the adenoviral delivery of human tissue inhibitors of matrix metalloproteinase (hTIMP)-1 and hTIMP-2 genes on the outcome of TNF/IFNgamma antitumor therapy. The dose of adenovirus was limited to 10(8) PFU per mouse owing to the additive toxicity of combining it with TNF/IFNgamma therapy. Nevertheless, this dose was sufficient to achieve highly efficient adenoviral transfer and expression of hTIMP-1 and hTIMP-2 in the liver, but not the tumor. Treatment with this low dose of AdhTIMP-1 or AdhTIMP-2 was not enough to protect the host against the toxic effects of TNF/IFNgamma. However, it was sufficient to show a synergistic effect of hTIMPs with TNF/IFNgamma such that tumors regressed significantly faster. Interestingly, only AdTIMP-2 was able to prevent relapses after treatment.

Suicide gene therapy using herpes simplex virus type-1 (HSV-1) thymidine kinase (TK) is a widely exploited approach for gene therapy of cancer and other hyperproliferative disorders. Despite its popularity, clinical success has been so far hampered mostly by the relative inefficiency of TK gene transfer and its limited bystander effect. Here we report that fusion of TK to an 11-amino-acid peptide from the basic domain of the HIV-1 Tat protein (Tat11) imparts cell membrane translocating ability to the enzyme and significantly increases its cytotoxic efficacy. In cells expressing Tat11-TK, this protein is found extracellularly, associated with cell surface heparan sulfate proteoglycans, and is released into the cell culture medium. Based on its interaction with HSPGs, the protein is then internalized by neighboring, nonexpressing cells, which become susceptible to cell death when treated with the nucleoside analogue acyclovir. As a consequence, co-cultures of wild-type cells with cells expressing Tat11-TK show increased sensitivity to ACV through a mechanism involving apoptosis. Modification of TK by fusion with Tat11 might constitute an important step for the optimization of TK suicide gene strategy for gene therapy of cellular proliferation.