高碘酸氧化肝素抑制P-选择素介导的A375细胞粘附

摘要通过选择性化学修饰制备出具有低抗凝血活性的高碘酸氧化肝素, 并系统地分析了其抗肿瘤细胞粘附的活性. 研究结果表明, 具有低抗凝血活性的高碘酸氧化肝素仍然可以显著地抑制P-选择素介导的人恶性黑素瘤A375细胞与血小板间的粘附.     

多发性骨髓瘤的骨髓形态学分析与临床应用

目的研究多发性骨髓瘤的骨髓形态学特征,探讨骨髓形态学在多发性骨髓瘤诊断中的临床应用价值。方法对广东医学院附属医院63例多发性骨髓瘤病人进行骨髓涂片及细胞染色和进行检验诊断分析。结果 94%的患者骨髓增生度都达到增生活跃以上,只有6%的是增生低下或极度低下,瘤细胞数量﹥15%,根据形态可分为四类;病人骨髓其他系包括巨核系、粒系、红系和淋巴系细胞都不同程度减少和受抑制。结论骨髓形态学分析是多发性骨髓瘤诊断和治疗疗效监测具有重要的临床应用价值,具有广泛开展的必要性和意义。     

Proteomic investigation of anti-tumor activities exerted by sinularin against A2058 melanoma cells

The extracts from soft corals have been increasingly investigated for biomedical and therapeutic purposes. The aim of this study is to examine and analyze the anti-tumor effects of the genus Sinularia extract sinularin on A2058 melanoma cells using MTT assay, cell migration assay, wound healing assay, flow cytometric analysis, and proteomic analysis. Sinularin dose-dependently (1-5 μg/mL) inhibited melanoma cell proliferation while the treatment at identical concentrations suppressed cell migration. Sinularin dose-dependently enhanced apoptotic melanoma cells and caused tumor cell accumulation at G2/M phase, indicating that sinularin exerts apoptosis-induced and cell cycle-delayed activities in A2058 melanoma cells. Comparative proteomic analysis was conducted to investigate the effects of sinularin at the molecular level by comparison between the protein profiling of melanoma cells treated with sinularin and without the treatment. Thirty-five differential proteins (13 upregulated and 22 downregulated) concerning the treatment were identified by liquid chromatography-tandem mass spectrometry. Proteomic data and Western blot displayed the levels of several tumor inhibitory or apoptosis-associated proteins including annexin A1, voltage-dependent anion-selective channel protein 1 and prohibitin (upregulated), heat shock protein 60, heat shock protein beta-1, and peroxiredoxin-2 (downregulated) in A2058 melanoma cells exposed to sinularin. Increased expression of p53, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, p21, and Bax and decreased expression of Bcl-2 in sinularin-treated melanoma cells suggest that the anti-tumor activities of sinularin against melanoma cells are particularly correlated with these pro-apoptotic factors. These data provide important information for the mechanisms of anti-tumor effects of sinularin on melanoma cells and may be helpful for drug development and progression monitoring of human melanoma.     

Association of Systemic Steroid Treatment and Outcome in Patients Treated with Immune Checkpoint Inhibitors: A Real-World Analysis

Background: Immune-related adverse events (irAEs) are inflammatory side effects, which can occur during immune-checkpoint(s) inhibitors (ICIs) therapy. Steroids are the first-line agents to manage irAEs because of their immunosuppressive properties. However, it is still debated whether or when steroids can be administered without abrogating the therapeutic efforts of immunotherapy. Methods: We retrospectively evaluated 146 patients with metastatic non-small cell lung cancer (NSCLC), melanoma and renal cell carcinoma (RCC) treated with ICIs. We assessed the progression-free survival (PFS) of patients treated with steroids due to an irAE compared to a no-steroid group. Results: The early treatment with steroid (within the first 30 days from the beginning of immunotherapy) was not related to a shorter PFS (p = 0.077). Interestingly, patients who were treated with steroids after 30 days from the start of immunotherapy had significantly longer PFS (p = 0.017). In a multivariate analysis, treatment with steroids after 30 days was an independent prognostic factor for PFS (HR: 0.59 [95% CI 0.36–0.97], p = 0.037). Conclusions: This retrospective study points out that early systemic steroids administration to manage irAEs might not have a detrimental effect on patient clinical outcome in NSCLC, melanoma and RCC patients.     

Structural and antitumor properties of the YSNSG cyclopeptide derived from tumstatin

We previously demonstrated that the NC1[alpha3(IV)185-191] CNYYSNS peptide inhibited in vivo tumor progression. The YSNS motif formed a beta turn crucial for biological activity. The aim of the present study was to design a YSNSG cyclopeptide with a constrained beta turn on the YSNS residues more stable than CNYYSNS. By nuclear magnetic resonance and molecular modeling, we demonstrated that the YSNSG cyclopeptide actually adopted the expected beta-turn conformation. It promoted melanoma cell adhesion and prevented their adhesion to the native peptide. It inhibited in vitro cell proliferation and migration through Matrigel by downregulating proteolytic cascades. Moreover, intraperitoneal administration of the YSNSG cyclopeptide inhibited melanoma progression far more efficiently than the native peptide. The increased solubility and stability at low pH of the YSNSG cyclopeptide suggest this peptide as a potent antitumor therapeutic agent.     

Methiothepin Increases Chemotherapy Efficacy against Resistant Melanoma Cells

We previously reported that methiothepin, a small molecule known as a nonselective serotonin 5-HT receptor antagonist, inhibited the doxorubicin efflux activity of the Hedgehog receptor Ptch1 and enhanced the cytotoxic, pro-apoptotic, anti-proliferative, and anti-clonogenic effects of doxorubicin on adrenocortical carcinoma cells. Here, we show that methiothepin also inhibits doxorubicin efflux and increases doxorubicin cytotoxicity in melanoma cells which endogenously overexpress Ptch1. Melanoma patients having the BRAF^V600E mutation are treated with vemurafenib, an inhibitor of BRAF^V600E, often in combination with trametinib, an inhibitor of MEK. Almost all patients ultimately acquire resistance to the treatment leading to disease progression. Here, we report that methiothepin overcomes the resistance of BRAF^V600E melanoma cells by enhancing the cytotoxicity of vemurafenib and trametinib on these cells leading to melanoma cells death. We observe that the addition of methiothepin to vemurafenib prevents migration of resistant melanoma cells more efficiently than vemurafenib alone. Our results provide an additional proof that Ptch1 drug efflux inhibition increases the effectiveness of anti-cancer treatments and overcomes resistance of melanoma cells expressing Ptch1.     

In vitro toxicity testing of zinc tetrasulfophthalocyanines in fibroblast and keratinocyte cells for the treatment of melanoma cancer by photodynamic therapy

A series of water-soluble tetrasulfonated metallophthalocyanines (MPcs) dyes have been studied to be used as a drug or photosensitizer (PS) in photodynamic therapy (PDT) for the treatment of cancers. During PDT the PS is administrated intravenously or topically to the patient before laser light at an appropriate wavelength is applied to the cancerous area to activate the PS. The activated PS will react with oxygen typically present in the cancerous tissue to generate reactive oxygen species for the destruction of the cancerous tissue. This in vitro study aimed at investigating the cytotoxic effects of different concentrations of zinc tetrasulfophthalocyanines (ZnTSPc) activated with a diode laser (λ = 672 nm) on melanoma, keratinocyte and fibroblast cells. To perform this study 3 × 10? cells/ml were seeded in 24-well plates and allowed to attach overnight, after which cells were treated with different concentrations of ZnTSPc. After 2h, cells were irradiated with a constant light dose of 4.5J/cm2. Post-irradiated cells were incubated for 24 h before cell viability was measured using the CellTiter-Blue Viability Assay. Data indicated high concentrations of ZnTSPc (60-100 μg/ml) in its inactive state are cytotoxic to the melanoma cancer cells. Also, results showed that photoactivated ZnTSPc (50 μg/ml) was able to reduce the cell viability of melanoma, fibroblast and keratinocyte cells to 61%, 81% and 83% respectively. At this photosensitizing concentration the efficacy the treatment light dose of 4.5J/cm2 against other light doses of 2.5J/cm2, 7.5J/cm2 and 10J/cm2 on the different cell lines were analyzed. ZnTSPc at a concentration of 50 μg/ml activated with a light dose of 4.5J/cm2 was the most efficient for the killing of melanoma cancer cells with reduced killing effects on healthy normal skin cells in comparison to the other treatment light doses. Melanoma cancer cells after PDT with a photosensitizing concentration of 50μg/ml and a treatment light dose of 4.5J/cm2 showed certain apoptosis characteristics such as chromatin condensation and fragmentation of the nucleus. This concludes that low concentrations of ZnTSPc activated with the appropriate light dose can be used to induce cell death in melanoma cells with the occurrence of minimal damage to surrounding healthy tissue.     

Catalytic immunotherapy-photothermal therapy combination for melanoma by ferroptosis-activating vaccine based on artificial nanoenzyme

Tumor cell vaccine is a promising approach for cancer therapy to activate tumor immune, which can be achieved by tumor cells immunogenic cell death (ICD), converting in situ tumors into endogenous vaccination strategy, and ferroptosis has been proved to induce ICD occurrence. Ferroptosis is triggered by artificial nanoenzyme copper telluride mimicking peroxidase and glutathione oxidase, based on which the ferroptosis-activating vaccine (termed as CM CTNPs@OVA) was designed and established for catalytic immunotherapy. Owing to photothermal effect of copper telluride, photothermal therapy (PTT) was combined for an intensive cancer therapeutic effect. CM CTNPs@OVA was composed of solid mesoporous copper telluride nanoparticles, ovalbumin (OVA) loaded in mesoporous, and melanoma cell membrane coating surface. In in vitro and in vivo investigations, CM CTNPs@OVA, with particle size of 113.7 ± 1.7 nm, was certified to release copper ions for ferroptosis initiation, and OVA directly maturated dendritic cell (DC) as exogenous antigens extracellularly. ICD was then induced by ferroptosis pathway and PTT to release damage-associated molecular patterns for DC maturation and subsequent T cells recruitment. CM CTNPs@OVA-treated melanoma with exited inhibition rate, proving that the strategy of catalytic immunotherapy-PTT combination by ferroptosis-activating vaccine possessed massive potential for melanoma therapy based on nanoenzyme copper telluride.     

ZnO‐Functionalized Upconverting Nanotheranostic Agent: Multi‐Modality Imaging‐Guided Chemotherapy with On‐Demand Drug Release Triggered by pH

Limited therapeutic efficiency and severe side effects in patients are two major issues existing in current chemotherapy of cancers in clinic. To design a proper theranostic platform seems thus quite needed to target cancer cells accurately by bioimaging and simultaneously release drugs on demand without premature leakage. A novel ZnO‐functionalized upconverting nanotheranostic platform has been fabricated for clear multi‐modality bioimaging (upconversion luminescence (UCL), computed tomography (CT), and magnetic resonance imaging (MRI)) and specific pH‐triggered on‐demand drug release. In our theranostic platform multi‐modality imaging provides much more detailed and exact information for cancer diagnosis than single‐modality imaging. In addition, ZnO can play the role of a “gatekeeper” to efficiently block the drug in the mesopores of the as‐prepared agents until it is dissolved in the acidic environment around tumors to realize sustained release of the drug. More importantly, the biodegradable ZnO, which is non‐toxic against normal tissues, endows the as‐prepared agents with high therapeutic effectiveness but very low side effects. These findings are of great interests and will inspire us much to develop novel effective imaging‐guided on‐demand chemotherapies in cancer treatment.     

Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia)

α‐Momorcharin (α‐MMC), a type I ribosome‐inactivating protein (RIP), has shown therapeutic potential such as anti‐tumor and anti‐viral agent. Traditional process of α‐MMC purification from bitter melon seeds was time consuming and low efficient. To take this challenge, we made an affinity matrix by coupling the monoclonal antibody (McAb) with Sepharose 4B. Using this attractive strategy, 196 mg of α‐MMC was obtained from 100 g of bitter melon seeds as the starting material. The yield of the protein was 2.7%. The homogeneity and properties of the protein were assessed by SDS‐PAGE, acidic PAGE, RP‐HPLC and N‐terminal sequence as well as Western blot. Purified α‐MMC showed remarkable inhibition to the melanoma cell line JAR and EMT‐62058. In addition, it also displayed obvious inhibition on hepatitis B virus (HBV). This work provided a simple, rapid and efficient approach for α‐MMC purification from Momordica charantia.     

Melanosomal damage in normal human melanocytes induced by UVB and metal uptake--a basis for the pro-oxidant state of melanoma

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.     

Virus-Like Particle-Encapsulated Doxorubicin Enters and Kills Murine Tumor Cells Differently from Free Doxorubicin

The use of nanoparticles as chemotherapeutic carriers has been suggested as a way to overcome a range of side effects associated with classical cancer treatment such as poor selectivity and tumor resurgence. Obtaining precise control of the size and shape of therapeutic nanoparticles is crucial to optimize the targeting of tumor sites. In this work, it is shown that a previously developed system of polypeptide encapsulating individual DNA molecules, that forms rod-shaped nanoparticles of precisely controlled aspect ratio, can be loaded with the DNA-intercalating chemotherapeutic drug doxorubicin (DOX). It is characterized the size and shape of the DOX loaded-Virus-Like DNA Particles (DOX-VLDP) and shown that in this system the DOX payload does not leak out. Through in vitro cell studies, it is shown that DOX-VLDP is internalized by melanoma tumor cells (B16F10 cells) in a delayed and endocytosis-dependent way culminating in increased cytotoxicity and selectivity to tumor cells in comparison with free DOX. In addition, it is found that DOX-VLDP trigger apoptosis and autophagy pathways in treated cells. Taken together, the data on the DOX-VLDP nanoparticles shows that they kill cancer cells differently from free DOX.     

Tricyclic analogues of epidithiodioxopiperazine alkaloids with promising in vitro and in vivo antitumor activity

Epipolythiodioxopiperazine (ETP) alkaloids are structurally elaborate alkaloids that show potent antitumor activity. However, their high toxicity and demonstrated interactions with various biological receptors compromises their therapeutic potential. In an effort to mitigate these disadvantages, a short stereocontrolled construction of tricyclic analogues of epidithiodioxopiperazine alkaloids was developed. Evaluation of a small library of such structures against two invasive cancer cell lines defined initial structure–activity relationships (SAR), which identified 1,4-dioxohexahydro-6H-3,8a-epidithiopyrrolo[1,2-a]pyrazine 3c and related structures as particularly promising antitumor agents. ETP alkaloid analogue 3c exhibits low nanomolar activity against both solid and blood tumors in vitro. In addition, 3c significantly suppresses tumor growth in mouse xenograft models of melanoma and lung cancer, without obvious signs of toxicity, following either intraperitoneal (IP) or oral administration. The short synthesis of molecules in this series will enable future mechanistic and translational studies of these structurally novel and highly promising clinical antitumor candidates.     

Quality by Design for Development,Optimization and Characterization of Brucine Ethosomal Gel for Skin Cancer Delivery

Natural products have been extensively used for treating a wide variety of disorders. In recent times, Brucine (BRU) as one of the natural medications extracted from seeds of nux vomica, was investigated for its anticancer activity. As far as we know, this is the first study on BRU anticancer activity against skin cancer. Thus, the rational of this work was implemented to develop, optimize and characterize the anticancer activity of BRU loaded ethosomal gel. Basically, thin film hydration method was used to formulate BRU ethosomal preparations, by means of Central composite design (CCD), which were operated to construct (3²) factorial design. Two independent variables were designated (phospholipid percentage and ethanol percentage) with three responses (vesicular size, encapsulation efficiency and flux). Based on the desirability function, one formula was selected and incorporated into HPMC gel base to develop BRU loaded ethosomal gel. The fabricated gel was assessed for all physical characterization. In-vitro release investigation, ex-vivo permeation and MTT calorimetric assay were performed. BRU loaded ethosomal gel exhibited acceptable values for the characterization parameters which stand proper for topical application. In-vitro release investigation was efficiently prolonged for 6 h. The flux from BRU loaded ethosome was enhanced screening optimum SSTF value. Finally, in-vitro cytotoxicity study proved that BRU loaded ethosomal gel significantly improved the anticancer activity of the drug against A375 human melanoma cell lines. Substantially, the investigation proposed a strong motivation for further study of the lately developed BRU loaded ethosomal gel as a prospective therapeutic strategy for melanoma treatment.     

Study of the development of chemoresistance in melanoma cell lines using proteome analysis

Malignant melanomas have poor prognosis since treatment with anti-neoplastic agents is mostly ineffective. The biological mechanisms of this strong intrinsic therapy resistance are unknown. In order to identify new molecular factors potentially associated with the drug-resistant phenotype of malignant melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to four commonly used anticancer drugs in melanoma treatment, i.e., vindesine, etoposide, cisplatin, and fotemustine, was characterized using proteomic tools (two-dimensional electrophoresis for protein fractionation and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry for protein identification). In the neutral and weak acidic milieu (pH 4.0-8.0) a total number of 14 proteins showed alterations in expression whereas 20 proteins were differentially expressed in the basic milieu (pH 8.0-11.0). Besides proteins with unknown physiologic function, several factors were identified that show chaperone activity. Moreover, proteins involved in drug detoxification, metabolism, and regulation of apoptotic pathways could be identified. The possible role of these proteins in the development of chemoresistance is discussed, although detailed functional tests with these proteins have still to be performed. Nevertheless, it is clear that this proteomic approach for studying chemoresistance phenomena is a prerequisite before further investigation can yield insight into the biology and development of drug resistance in malignant melanoma.     

Latitude and incidence of ocular melanoma

We investigated the associations between latitude and the incidence of two different types of ocular melanoma, external ocular melanoma (exposed to sunlight) and internal melanoma (not exposed to sunlight), separately. Using 1992-2002 data from the Surveillance, Epidemiology, and End Results (SEER) Program of National Cancer Institute, we identified 2142 ocular melanoma cases in non-Hispanic whites, and then regressed the incidences of various types of ocular melanomas with latitude. Our analysis indicated that the higher the latitude (away from the equator, the less sun exposure), the lower the risk of external ocular melanoma (eyelid and conjunctival melanomas) among non-Hispanic whites (P for trend = 0.018). The incidence increased 2.48 fold from 47-48 degrees to 20-22 degrees. This trend is very similar to that of skin melanoma. The incidence of internal ocular melanoma (uveal melanoma) increased significantly with increasing latitudes (the less sun exposure, P for trend < 0.0001), it increased 4.91 fold from 20-22 degrees to 47-48 degrees. The latitudinal patterns of ocular melanomas may reflect the dual effects of sunlight exposure, i.e. a mutagenic effect of direct solar radiation on external ocular melanomas and a protective effect for internal uveal melanoma, which is similar to the sun radiation protective effects for various internal malignant tumors that are not exposed to the sunlight.     

Synthesis and characterization of a cobalamin-colchicine conjugate as a novel tumor-targeted cytotoxin

Colchicine was derivatized at C7 with p-alkoxyacetophenone and conjugated to cobalamin (vitamin B(12)) through an acid-labile hydrazone linker. The cobalamin moiety leads to preferential uptake of the cobalamin-colchicine prodrug by cancer cells, whereupon the hydrazone linker undergoes hydrolysis in the lysosome to unmask colchicine, which acts as a potent cytotoxin by stabilizing microtubules and causing cell death. The bioconjugate is stable in cell culture media and at neutral pH but undergoes hydrolysis with a half-life of 138 min at pH 4.5. The colchicine-cobalamin bioconjugate exhibits nanomolar LC(50) values against breast, brain, and melanoma cancer cell lines in culture. Attachment of colchicine to cobalamin is expected to increase the therapeutic index of the drug by limiting the side effects caused by the current nonselective administration of tubulin-targeted chemotherapeutic drugs.