Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Di-(2-ethylhexyl) phthalate-induced tumor growth is regulated by primary cilium formation via the axis of H2O2 production-thymosin beta-4 gene expression.

Di-(2-ethylhexyl) phthalate (DEHP) that is one of the most commonly used phthalates in manufacturing plastic wares regulates tumorigenesis. Thymosin beta-4 (TB4), an actin-sequestering protein, has been reported as a novel regulator to form primary cilia that are antenna-like organelles playing a role in various physiological homeostasis and pathological development including tumorigenesis. Here, we investigated whether DEHP affects tumor growth via primary cilium (PC) formation via the axis of TB4 gene expression and the production of reactive oxygen species (ROS). Tumor growth was increased by DEHP treatment that enhanced TB4 expression, PC formation and ROS production. The number of cells with primary cilia was enhanced time-dependently higher in HeLa cells incubated in the culture medium with 0.1% fetal bovine serum (FBS). The number of cells with primary cilia was decreased by the inhibition of TB4 expression. The incubation of cells with 0.1% FBS enhanced ROS production and the transcriptional activity of TB4 that was reduced by ciliobrevin A (CilioA), the inhibitor of ciliogenesis. ROS production was decreased by catalase treatment but not by mito-TEMPO, which affected to PC formation with the same trend. H2O2 production was reduced by siRNA-based inhibition of TB4 expression. H2O2 also increased the number of ciliated cells, which was reduced by siRNA-TB4 or the co-incubation with CilioA. Tumor cell viability was maintained by ciliogenesis, which was correlated with the changes of intracellular ATP amount rather than a simple mitochondrial enzyme activity. TB4 overexpression enhanced PC formation and DEHP-induced tumor growth. Taken together, data demonstrate that DEHP-induced tumor growth might be controlled by PC formation via TB4-H2O2 axis. Therefore, it suggests that TB4 could be a novel bio-marker to expect the risk of DEHP on tumor growth.

Regulatory T Cells Play an Important Role in the Prevention of Murine Melanocytic Nevi and Melanomas.

Melanocytic nevi are benign proliferations of pigment cells that can occasionally develop into melanomas. There is a significant correlation between increased nevus numbers and melanoma development. Our previous reports revealed that 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced dysplastic nevi in C3H/HeN mice, with a potential to transform into melanomas. To understand the immune mechanisms behind this transformation, we applied increasing DMBA doses followed by TPA to the skin of C3H/HeN mice. We observed that increased doses of DMBA correlated well with increased numbers of nevi. The increased DMBA dose induced diminished immune responses and promoted the expansion of regulatory T cells (Treg) that resulted in increased IL10 and reduced IFNγ levels. Mice with increased nevus numbers had loss of p16 expression. These mice had increased migration of melanocytic cells to lymph nodes (LN) and a greater percent of LNs produced immortalized melanocytic cell lines. DMBA-induced immunosuppression was lost in CD4-knockout (KO) mice. Lymphocytes in the CD4KO mice produced less IL10 than CD8KO mice. Furthermore, CD4KO mice had significantly reduced nevus numbers and size compared with wild-type and CD8KO mice. These results suggest that Tregs play a vital role in the incidence of nevi and their progression to melanoma.Prevention Relevance: There has been little progress in developing novel strategies for preventing premalignant dysplastic nevi from becoming melanomas. In this study in mice, regulatory-T cells enhanced progression of benign nevi to malignant melanomas; and by inhibiting their activity, melanomas could be retarded. The findings identify new possibilities for melanoma prevention in high risk individuals.

Isolation and Characterization of Innate Lymphoid Cells within the Murine Tumor Microenvironment.

Innate lymphoid cells (ILCs) are important for both tissue immunity and tissue homeostasis. They are classified into three groups: Group 1 ILCs include NK cells, which are important in eliciting immunity against intracellular pathogens; group 2 ILCs protect against parasitic helminths; and group 3 ILCs protect against extracellular pathogens. The role of ILCs in cancer immunity remains unclear. In this chapter, we discuss methods for isolating and characterizing tumor-infiltrating ILC subsets within the tumor microenvironment in an experimental murine model of B16 melanoma. The chapter also highlights the expression of PD-1 on the various ILC subsets within the tumor microenvironment.

Amine derivatives of furocoumarin induce melanogenesis by activating Akt/GSK-3ß/ß-catenin signal pathway.

BACKGROUND: Melanogenesis, or the biosynthesis of melanin, plays a critical role in the pigmentation of skin, hair, and eyes. Reduced melanogenesis may lead to depigmentation conditions such as vitiligo. Psoralen, a furocoumarin derivative, is closely associated with melanogenesis, and its derivative 8-methoxypsoralen is used in psoralen and ultraviolet A therapy for pigmentation disorders. In a previous study, we synthesized a new series of amine derivatives of furocoumarin, of which 5-(morpholinomethyl)-3-phenyl-7H-furo[3,2-g]chromen-7-one (encoded as D206008) showed a remarkable melanogenic effect in B16 murine cells. METHODS: In this study, we examined the effects of D206008 on the melanogenesis-related pathways in B16 cells. D206008 increased melanin production and tyrosinase (TYR) activity, as well as the mRNA and protein expression levels of the melanogenic enzymes TYR, TRP-1 and TRP-2, and the melanogenesis-related transcription factor microphthalmia-associated transcription factor (MITF) in a dose-dependent (0-100 µM) and time-dependent (0-48 hours) manner. RESULTS: Mechanistically, D206008 inhibited ß-catenin degradation by enhancing the phosphorylation of Akt and glycogen synthase kinase-3ß (GSK-3ß), which increased the accumulation of ß-catenin in the cytoplasm. Nuclear translocation of ß-catenin also increased in response to D206008 treatment. CONCLUSION: Taken together, these data indicate that D206008 promotes melanin synthesis by stimulating the nuclear translocation of ß-catenin, which activates MITF transcription and eventually melanogenesis.

KLF9-dependent ROS regulate melanoma progression in stage-specific manner.

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten-/- primary melanomas. It also, as expected, promoted BrafCA/Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

The Ashitaba (Angelica keiskei) Chalcones 4-hydroxyderricin and Xanthoangelol Suppress Melanomagenesis By Targeting BRAF and PI3K.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607-20. ©2018 AACR.

Angiotensin II promotes pulmonary metastasis of melanoma through the activation of adhesion molecules in vascular endothelial cells.

Hypertension is considered as one of the cancer progressive factors, and often found comorbidity in cancer patients. Renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure, and angiotensin II (Ang II) is well known pressor peptide associated with RAS. Ang II has been reported to accelerate progression and metastasis of cancer cells. However, its precise mechanisms have not been fully understood. In this study, we sought to elucidate the mechanisms by which Ang II exacerbates hematogenous metastasis in mouse melanoma cells, focusing the adhesion pathway in vascular endothelial cells. For this purpose, B16/F10 mouse melanoma cells, which do not express the Ang II type 1 receptor (AT1R), were intravenously injected into C57BL/6 mice. Two weeks after cell injection, the number of lung metastatic colonies was significantly higher in the Ang II-treated group (1 µg/kg/min) than in the vehicle-treated group. The AT1R blocker valsartan (40 mg/kg/day), but not the calcium channel blocker amlodipine (5 or 10 mg/kg/day), significantly suppressed the effect of Ang II. In endothelium-specific Agtr1a knockout mice, Ang II-mediated acceleration of lung metastases of melanoma cells was significantly diminished. Ang II treatment significantly increased E-selectin mRNA expression in vascular endothelial cells collected from lung tissues, and thus promoted adherence of melanoma cells to the vascular endothelium. Ang II-accelerated lung metastases of melanoma cells were also suppressed by treatment with anti-E-selectin antibody (20 mg/kg). Taken together, Ang II-treatment exacerbates hematogenous cancer metastasis by promoting E-selectin-mediated adhesion of cancer cells to vascular endothelial cells.

Impact of Chemical-Induced Mutational Load Increase on Immune Checkpoint Therapy in Poorly Responsive Murine Tumors.

A recurring historic finding in cancer drug development is encouraging antitumor effects observed in tumor-bearing mice that fail to translate into the clinic. An intriguing exception to this pattern is immune checkpoint therapy, as the sustained tumor regressions observed in subsets of cancer patients are rare in mice. Reasoning that this may be due in part to relatively low mutational loads of mouse tumors, we mutagenized transplantable mouse tumor cell lines EMT-6/P, B16F1, RENCA, CT26, and MC38 in vitro with methylnitro-nitrosoguanidine (MNNG) or ethylmethane sulfonate (EMS) and tested their responsiveness to PD-L1 blockade. Exome sequencing confirmed an increase in somatic mutations by mutagen treatment, an effect mimicked in EMT-6 variants chronically exposed in vivo to cisplatin or cyclophosphamide. Certain mutagenized variants of B16F1, EMT-6/P, CT26, and MC38 (but not RENCA) were more immunogenic than their parents, yet anti-PD-L1 sensitization developed only in some EMT-6/P and B16F1 variants. Treatment response patterns corresponded with changes in immune cell infiltration and especially increases in CD8+ T cells. Chronically cisplatin-exposed EMT-6 variants were also more responsive to anti-PD-L1 therapy. Although tumor PD-L1 expression was upregulated in in vivo chemotherapy-exposed variants, PD-L1 expression levels were not consistently associated with anti-PD-L1 treatment activity across mutagenized or chemotherapy-exposed variants. In summary, mutagenized and more immunogenic mouse tumors were not universally sensitized to PD-L1 blockade. Chemically mutagenized variants may be useful to evaluate the impact of immunologically "hot" or "cold" tumors with a high mutational load, to which certain chemotherapy agents may contribute, on immunotherapy outcomes. Mol Cancer Ther; 17(4); 869-82. ©2018 AACR.

Nonylphenol increases tumor formation and growth by suppressing gender-independent lymphocyte proliferation and macrophage activation.

Nonylphenol (NP) is a well-known endocrine disruptor that influences sexual and reproductive development. Here, we investigated whether NP affects immune responses that are associated with tumor initiation and progression. When spleen cells were incubated with lipopolysaccharide (LPS) and concanavalin A in the presence of 10-4 M NP, the proliferation of B and T lymphocytes was reduced compared with that in controls, in a gender-independent fashion. While 10-4 M NP also decreased the production of nitric oxide (NO) in LPS-stimulated bone marrow-derived macrophages (BMDMs), no changes in NO production were detected following treatment with 10-5 M NP. LPS-stimulated expression of iNOS, COX2, IL-6 and TNF-α in BMDMs was reduced after 6 or 18 hours of incubation with 10-5 M NP. Furthermore, when mice were pre-exposed to NP for 7 days prior to the injection of B16F10 melanoma cells, the rates of tumor nodule formation and relative tumor growth were higher than those in the control group. In vivo immunosuppressive effect was also clarified by the inhibition of proliferation in B/T lymphocyte and cytokine production in peritoneal macrophages from the mice pretreated with NP for 7 days. Taken together, these data demonstrate that NP could affect the immune responses of lymphocytes and macrophages, leading to the suppression of their tumor-preventing ability. This suggests that individuals at high risk for tumor development should avoid frequent exposure to NP and other endocrine disruptors.

Chaetocin inhibits IBMX-induced melanogenesis in B16F10 mouse melanoma cells through activation of ERK.

Chaetocin is a natural product isolated from Chaetomium species that has anti-bacterial and anti-myeloma activities. In this study, we investigated the inhibitory effect of chaetocin on melanogenesis and the underlying mechanisms in B16F10 mouse melanoma cells. In the present study, chaetocin significantly inhibited IBMX-induced melanin production and tyrosinase activity without any cytotoxicity. Furthermore, chaetocin down-regulated both the protein and mRNA levels of tyrosinase, which is a specific enzyme that catalyzes the conversion of tyrosine to melanin. We also observed that the protein level of MITF was significantly reduced by chaetocin treatment. In addition, we found that the anti-melanogenic effect of chaetocin was suppressed by treatment with the specific ERK inhibitor (PD98059). Accordingly, chaetocin inhibited melanogenesis via suppressing the protein level of MITF followed by activation of the ERK signaling pathway. These data suggest that chaetocin may be a potential anti-melanogenic agent for use in skin-whitening cosmetics and a topical agent for treatment of hyperpigmentation disorders.

Effect of novel cyclohexane diester and benzene diester derivatives on melanogenesis.

In order to investigate potent whitening agents, we synthesized 15 cyclohexane diester derivatives and 15 benzene diester derivatives. To evaluate their structure-cytotoxicity relationships, we performed cell cytotoxicity tests on B16F10 mouse melanoma cells. To understand their whitening effects, melanin synthesis inhibitory activities in B16F10 cells and mushroom tyrosinase inhibitory activities were performed. In most cases, cell cytotoxicity was observed to be lower in 1,3-diester than in 1,2- and 1,4-diesters; when it came to the structural isomer of the side chain, all derivatives except the 1,2-cyclohexane diester derivatives showed lower cell cytotoxicity in the branch type of the side chain than in the linear type. Among the compounds evaluated, the compounds cyclohexane-1,3-diyl bis(decanoate), cyclohexane-1,4-diyl dioctanoate, and 1,3-phenylene bis (2-ethylhexanoate) emerged as potent melanin synthesis inhibitors. Our goal was to determine the expression levels of proteins involved in melanogenesis, Western blotting and RT-PCR showing that these compounds decreased tyrosinase, TRP-1, and TRP-2 while demonstrating significantly low cytotoxicity.

Pathways of tumor development and progression in drug-induced nonmelanoma skin cancer: a new hope or the next great confusion?

The factors that lead to the clinical manifestation of the nonmelanocytic skin tumors are different. Ultraviolet radiation, infections with human papillomaviruses, and inherited or iatrogenic-induced immunosuppression (in cases of autoimmune diseases and organ transplant recipients) are considered to be some of the most important generators and/or costimulating factors supporting the appearance of "de-novo" mutations and obstruct, in one or another way, the cell cycle arrest, the programmed cell death (apoptosis), and the immunosurveillance. Preconditions are thus created for the initial persistence and subsequent proliferation of the malignant cell branch in the genome, with the simultaneous increase of the risk of nonmelanocytic skin tumor manifestation.A number of medical drugs that possess a currently well-known selective, targeting, and immunomodulating effect, like the TNF-alpha inhibitors for example, most probably possess an additional blocking action on the death receptors within the framework of the extrinsic apoptotic pathway. In this way, they seem to be one of the major factors for the clinical manifestation not only of nonmelanocytic skin but also of a number of other type of tumors with a dependency on the genetic predisposition of each separate patient.This article focuses the attention on the basic exogenic and endogenic factors that affect the regulatory processes of the cellular cycle, apoptosis, immunosurveillance, and the human inflammasome in patients with nonmelanocytic skin tumors. These processes are interwoven in a complex network and are controlled by (1) the genome regulator p53, (2) its interaction with the proapoptotic acting proteins Bak and Bax, (3) as well as the interaction with the key regulatory protein of the inflammasome-ASC/TMS1.As a process, the malignant transformation is exceptionally dynamic, plastic, and adaptive. The exterior "interferences", on the part of the clinician, in the form of a planned therapy should be targeted at the simultaneous impact on the various pathogenetic chains with the objective of bringing the tumor cells to their total collapse. This can be made possible only after the careful and simultaneous-or parallel-examination of a much greater number of markers that serve to characterize the process of the malignant transformation-a fact, which is currently being disregarded by many researchers.

Antisense oligonucleotides against TNFR1 prevent toxicity of TNF/IFNγ treatment in mouse tumor models.

Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and toxic effects because both of them are absent in P55-deficient mice. In previous work we demonstrated that P55+/- mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNγ) remain fully functional in these mice. Hence, a high dose of TNF/IFNγ has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNγ treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNγ antitumor therapy.

Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells

The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system.B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed.BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.

A novel form of melanoma apoptosis resistance: melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death.

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.

Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genes implicated in human melanoma.

The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.

Synthesis and biological evaluation of 125I-erythropoietin as a potential radiopharmaceutical agent for tumours

Erythropoietin (EPO) is a glycoprotein hormone responsible for regulating erythropoiesis. Expression of EPO and EPO receptors (EPOr) has recently been demonstrated in some neoplastic cell lines and tumours, suggesting a potential new target for therapy. In this work, EPO was labeled with iodine-125 using the lactoperoxidase method, known to prevent damage to protein during radioiodination, and labeling conditions were optimized. In vitro stability studies have shown that 125I-EPO is radiochemically stable for 20 days after radiolabeling. In vitro cell binding studies have demonstrated very low binding (<2 percent) of EPO to normal and neoplastic cell lines tested. As expected, the biodistribution in healthy mice exhibited comparatively high rates of fixation in the organs of the excretory system. Thyroid also proved to be a critical organ which may indicate in vivo dissociation of 125I-EPO. In mice with induced melanoma, only a residual fixation in the tumour was evident. Further studies are warranted on other tumoral cell lines to better understand the binding process and internalization into cells. Studies on EPO labeled with carbon-11 could be valuable, since there is a greater chance of preserving the biological activity of the protein using this method.

A eritropoetina (EPO) é um hormônio glicoprotéico responsável pela regulação da eritropoese. Recentemente foi demonstrado que os receptores de EPO (EPOr) estão expressos em algumas linhas celulares neoplásicas, o que sugere a sua potencialidade como um novo alvo terapêutico. Neste trabalho a EPO foi radiomarcada com iodo-125 através do método da lactoperoxidase, menos agressivo para a viabilidade biológica das proteínas. A 125I-EPO revelou ser radioquimicamente estável durante 20 dias após a síntese. Um estudo biológico in vitro em linhas celulares tumorais demonstrou que a 125I-EPO apresenta uma ligação muito fraca (<2 por cento), tanto em células normais como nas linhagens tumorais testadas. A biodistribuição em camundongos saudáveis apresentou taxas de fixação relativamente maiores nos órgãos excretores e a tireóide revelou ser o órgão crítico, o que pode indicar a dissociação in vivo da 125I-EPO. No estudo em camundongos com melanoma induzido a fixação no tumor foi residual. Serão, no entanto, necessários novos estudos em outras linhagens tumorais para entender o seu processo de internalização e ligação nas células. Estudos da EPO radiomarcada com carbono-11 poderão também revelar-se interessantes, já que neste método há maior probabilidade da atividade biológica ser preservada.

An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles.

Here, we investigated whether titanium dioxide (TiO2) nanoparticles affect in vivo tumor growth through the modulation of mononuclear leukocytes. In vitro lymphocyte proliferation by lipopolysaccharide (LPS) or concanavalin A (ConA) was reduced by < 25 nm TiO2 with a dose-dependent manner. Similarly, TiO2 nanoparticles inhibited nitric oxide (NO) production from bone marrow-derived macrophages obtained from naïve mice. When mice were intraperitoneally (IP) injected with < 25 or < 100 nm TiO2 once a day for 7 days, total cell number of splenocytes was reduced in the spleen of TiO2 nanoparticle-exposed mice. Both CD4+ and CD8+ T-lymphocyte numbers were significantly decreased and B-lymphocyte development was retarded by host exposure to the TiO2 nanoparticles. LPS-stimulated spleen cell proliferation was significantly reduced by host exposure to < 25 or < 100 nm TiO2, but no changes were detected in ConA-stimulated spleen cell proliferation. Further, LPS-stimulated cytokine production by peritoneal macrophages and the percentage of NK1.1+ natural killer cells among splenocytes was reduced by the host exposures to the TiO2 nanoparticles. When mice were IP injected with TiO2 nanoparticles once a day for 28 days prior to the subcutaneous implantation of B16F10 melanoma cells, tumor growth was subsequently significantly increased. Collectively, these results show that TiO2 nanoparticles may damage the development and proliferation of B- and T-lymphocytes, reduce the activity of macrophages, and decrease natural killer (NK) cell population levels, outcomes that appear to lead to an increase in tumor growth in situ. These studies allow us to suggest that TiO2 nanoparticles might have the potential to enhance tumor growth through immunomodulation of B- and T-lymphocytes, macrophages, and NK cells.

Preformulation, formulation, and in vivo efficacy of topically applied apomine.

Apomine is a novel compound that inhibits the mevalonate/isoprenoid pathway of cholesterol synthesis and may prove effective as a skin cancer chemoprevention therapy. This research was focused on the development of a new delivery approach for chemoprevention of melanoma using topically delivered apomine. This included evaluating the effect of several factors on the stability of apomine in solution, utilizing these to develop a topical formulation, and conducting efficacy studies with the developed topical formulation in the TPras mouse model. Preformulation included the influence of pH, buffer species, ionic strength, and organic solvents on the stability of apomine at four different temperatures. Apomine was determined to undergo apparent first-order degradation kinetics for all conditions evaluated. Apomine undergoes base-catalyzed degradation. Less than 15% degradation is observed after >200 days under acidic conditions. Long-term stability studies were performed on two different topical cream formulations and indicate that both formulations are chemically stable for over 1 year at both 4 and 23 degrees C. The efficacy of topically applied apomine, from ethanol and developed 1% cream, was evaluated in vivo against the incidence of melanoma. Regardless of delivery vehicle apomine treatment caused a significant reduction in tumor incidence. Ethyl alcohol application of apomine resulted in a greater tumor incidence reduction when compared to the development cream formulation; however, this difference was not significant.