Nanoparticulate delivery of potent microtubule inhibitor for metastatic melanoma treatment.
ABSTRACT: Melanoma is the most aggressive type of skin cancer, which readily metastasizes through lymph nodes to the lungs, liver, and brain. Since the repeated administration of most chemotherapeutic drugs develops chemoresistance and severe systemic toxicities, herein we synthesized 2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) methanone (abbreviated as QW-296), a novel tubulin destabilizing agent with little susceptible to transporter-mediated drug resistance. QW-296 disturbed the microtubule dynamics at the nanomolar concentration in A375 and B16F10 melanoma cells. QW-296 binding to colchicine-binding site on tubulin protein was confirmed by molecular modeling and tubulin polymerization assay. QW-296 significantly inhibited A375 and B16F10 cell proliferation, induced G2/M cell cycle arrest and led to apoptosis and cell death. To improve its aqueous solubility, QW-296 was encapsulated into methoxy poly(ethyleneglycol)-b-poly(carbonate-co-lactide) [mPEG-b-P(CB-co-LA)] polymeric nanoparticles by solvent evaporation, with the mean particle size of 122.0?±?2.28?nm and drug loading of 3.70% (w/w). Systemic administration of QW-296 loaded nanoparticles into C57/BL6 albino mice bearing lung metastatic melanoma at the dose of 20?mg/kg 4 times a week for 1.5?weeks resulted in significant tumor regression and prolonged mouse median survival without significant change in mouse body weight. In conclusion, QW-296 loaded nanoparticles have the potential to treat metastatic melanoma.
Project description:Castration-resistant prostate cancer that has become resistant to docetaxel (DTX) represents one of the greatest clinical challenges in the management of this malignancy. There is an urgent need to develop novel therapeutic agents to overcome chemoresistance and improve the overall survival of patients. We have designed a novel microtubule destabilizer (2-(4-hydroxy-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (QW-296) and combined it with a newly synthesized hedgehog (Hh) signaling pathway inhibitor 2-chloro-N 1-[4-chloro-3-(2-pyridinyl)phenyl]-N 4,N 4- bis(2-pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5) to treat taxane-resistant (TXR) prostate cancer. The combination of QW-296 and MDB5 exhibited stronger anticancer activity toward DU145-TXR and PC3-TXR cells and suppressed tumor colony formation when compared with single-drug treatment. Because these drugs are hydrophobic, we synthesized the mPEG-p(TMC-MBC) [methoxy-poly(ethylene glycol)-block-poly(trimethylene carbonate-co-2-methyl-2-benzoxycarbonyl-propylene carbonate)] copolymer, which could self-assemble into micelles with loading capacities of 8.13% ± 0.75% and 9.12% ± 0.69% for QW-296 and MDB5, respectively. Further, these micelles provided controlled the respective drug release of 58% and 42% release of QW-296 and MDB5 within 24 hours when dialyzed against PBS (pH 7.4). We established an orthotopic prostate tumor in nude mice using stably luciferase expressing PC3-TXR cells. There was maximum tumor growth inhibition in the group treated with the combination therapy of QW-296 and MDB5 in micelles compared with their monotherapies or combination therapy formulated in cosolvent. The overall findings suggest that combination therapy with QW-296 and MDB5 has great clinical potential to treat TXR prostate cancer, and copolymer mPEG-p(TMC-MBC) could serve as an effective delivery vehicle to boost therapeutic efficacy in vivo.
Project description:Background:Melanoma is the most common symptom of aggressive skin cancer, and it has become a serious health concern worldwide in recent years. The metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options. Effective yet safe therapeutic options are still lacking. Alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma. This study aimed to develop albumin nanoparticles (ANPs) coated with macrophage plasma membranes (RANPs) loaded with paclitaxel (PTX) to achieve targeted therapy against malignant melanoma. Methods:Membrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Albumin nanoparticles were prepared through a well-studied nanoprecipitation process. At last, the two components were then coextruded through a 200 nm porous polycarbonate membrane. Results:Using paclitaxel as the model drug, PTX-loaded RANPs displayed significantly enhanced cytotoxicity and apoptosis rates compared to albumin nanoparticles without membrane coating in the murine melanoma cell line B16F10. RANPs also exhibited significantly higher internalization efficiency in B16F10 cells than albumin nanoparticles without a membrane coating. Next, a B16F10 tumor xenograft mouse model was established to explore the biodistribution profiles of RANPs, which showed prolonged blood circulation and selective accumulation at the tumor site. PTX-loaded RANPs also demonstrated greatly improved antitumor efficacy in B16F10 tumor-bearing mouse xenografts. Conclusion:Albumin-based nanoscale delivery systems coated with macrophage plasma membranes offer a highly promising approach to achieve tumor-targeted therapy following systemic administration.
Project description:The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented.Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay.The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis.These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy.
Project description:A herbal formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos was traditionally used to treat melanoma. Constitutively active signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target in melanoma. Here we investigated whether an ethanolic extract of SL (SLE) exerted anti-melanoma activities by inhibiting STAT3 signaling. B16F10 allograft model, A375 and B16F10 cells were employed to assess the in vivo and in vitro anti-melanoma activities of SLE. A375 cells stably expressing STAT3C, a constitutively active STAT3 mutant, were used to determine the role of STAT3 signaling in SLE's anti-melanoma effects. Intragastric administration of SLE (1.2?g/kg) potently inhibited melanoma growth in mice and inhibited STAT3 phosphorylation in the tumors. In cultured cells, SLE dramatically reduced cell viability, induced apoptosis, suppressed migration and invasion, and restrained STAT3 activation and nuclear localization. STAT3C overexpression in A375 cells diminished SLE's effects on cell viability, apoptosis and invasion. Collectively, SLE exerted potent anti-melanoma effects partially by inhibiting STAT3 signaling. This study provides pharmacological justification for the traditional use of this formula in treating melanoma, and suggests that SLE has the potential to be developed as a modern alternative and/or complimentary agent for melanoma treatment and prevention.
Project description:Melanoma, as the most aggressive and treatment-resistant skin malignancy, is responsible for about 80% of all skin cancer mortalities. Prone to invade into the dermis and form distant metastases significantly reduce the patient survival rate. Therefore, early treatment of the melanoma in situ or timely blocking the deterioration of metastases is critical. In this study, a sulfur dioxide (SO2) polymer prodrug was designed as both an intracellular glutathione (GSH)-responsive SO2 generator and a carrier of doxorubicin (DOX), and used for the treatment of subcutaneous and metastatic melanoma. Firstly, chemical conjugation of 4-N-(2,4-dinitrobenzenesulfonyl)-imino-1-butyric acid (DIBA) onto the side chains of methoxy poly (ethylene glycol) grafted dextran (mPEG-g-Dex) resulted in the synthesis of the amphiphilic polymer prodrug of SO2, mPEG-g-Dex (DIBA). The obtained mPEG-g-Dex (DIBA) could self-assemble into stable micellar nanoparticles and exhibited a glutathione-responsive SO2 release behavior. Subsequently, DOX was encapsulated into the core of mPEG-g-Dex (DIBA) micelles to form DOX-loaded nanoparticles (PDDN-DOX). The formed PDDN-DOX could be internalized by B16F10 cells and synchronously release DOX and SO2 into the tumor cells. As a result, PDDN-DOX exerted synergistic anti-tumor effects in B16F10 melanoma cells because of the oxidative damage properties of SO2 and toxic effects of DOX. Furthermore, in vivo experiments verified that PDDN-DOX had great potential for the treatment of subcutaneous and metastasis melanoma. Collectively, our present work demonstrates that the combination of SO2-based gas therapy and chemotherapeutics offers a new avenue for inhibiting melanoma progression and metastases. Graphical abstract Image 1 Highlights • A novel SO2 polymer prodrug is prepared and used as both a GSH-responsive SO2 generator and a carrier of DOX.• The DOX-loaded SO2 polymer prodrug nanoparticles (termed PDDN-DOX) can simultaneously release SO2 and DOX in response to GSH.• The PDDN-DOX demonstrates synergistic anticancer effect in B16F10 melanoma cells.• The PDDN-DOX is promising for the treatment of subcutaneous and metastatic melanoma.
Project description:Focal adhesion kinase (FAK) is involved in tumor cell migration and metastasis. However, the underlying mechanism remains unclear. Here, we present a signaling pathway involved in the regulation of melanoma cell migration by FAK. We found that the interference of FAK expression suppressed B16F10 cell migration/metastasis, and altered the expressions of genes involved in melanoma migration/metastasis. The down-regulation of FAK inhibited the expression of p-SrcY416, p-ERK1/2, Stat3 and p-Stat3Y705, while promoted the expression of PPAR?, miR-125b and E-cadherin. Then we found that FAK inhibited E-cadherin expression via p-SrcY416/p-ERK1/2/ p-Stat3Y705 and PPAR?/miR-125b/Stat3 signaling pathway in B16F10 cell. Moreover, miR-125b inhibited B16F10 cell migration. Furthermore, we repeated the key data with human melanoma cell line A375. The results obtained from A375 cells fell in line with those from B16F10 cells. Using Oncomine database, we found that the mRNA levels of FAK, Src, ERK1/2 and Stat3 increased, while the mRNA levels of PPAR?, C21orf34 (miR-125b host gene) and E-cadherin decreased in human metastatic melanoma. The data from human breast cancer confirmed those from metastatic melanoma.Taken together, our study suggests that down-regulation of FAK promotes E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPAR?/miR-125b/Stat3 signaling pathway. Our findings provide a novel explanation regarding how FAK promotes melanoma cell migration, suggesting that FAK might be a potential target for melanoma therapy.
Project description:Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are crucial targetable enzymes in cancer management. Therefore, herein, new 2-[(5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)thio]-N-(thiazol/benzothiazol-2-yl)acetamides (2a-i) were designed and synthesized as EGFR and COX-2 inhibitors. The cytotoxic effects of compounds 2a-i on HCT116 human colorectal carcinoma, A549 human lung adenocarcinoma, and A375 human melanoma cell lines were determined using MTT assay. 2-[(5-((1H-Indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)thio]-N-(6-ethoxybenzothiazol-2-yl)acetamide (2e) exhibited the most significant anticancer activity against HCT116, A549, and A375 cell lines with IC50 values of 6.43 ± 0.72 ?M, 9.62 ± 1.14 ?M, and 8.07 ± 1.36 ?M, respectively, when compared with erlotinib (IC50 = 17.86 ± 3.22 ?M, 19.41 ± 2.38 ?M, and 23.81 ± 4.17 ?M, respectively). Further mechanistic assays demonstrated that compound 2e enhanced apoptosis (28.35%) in HCT116 cells more significantly than erlotinib (7.42%) and caused notable EGFR inhibition with an IC50 value of 2.80 ± 0.52 ?M when compared with erlotinib (IC50 = 0.04 ± 0.01 ?M). However, compound 2e did not cause any significant COX-2 inhibition, indicating that this compound showed COX-independent anticancer activity. The molecular docking study of compound 2e emphasized that the benzothiazole ring of this compound occupied the allosteric pocket in the EGFR active site. In conclusion, compound 2e is a promising EGFR inhibitor that warrants further clinical investigations.
Project description:Garcinol (GAR) is a naturally occurring polyisoprenylated phenolic compound. It has been recently investigated for its biological activities such as antioxidant, anti-inflammatory, anti ulcer, and antiproliferative effect on a wide range of human cancer cell lines. Though the outcomes are very promising, its extreme insolubility in water remains the main obstacle for its clinical application. Herein we report the formulation of GAR entrapped PLGA nanoparticles by nanoprecipitation method using vitamin E TPGS as an emulsifier. The nanoparticles were characterized for size, surface morphology, surface charge, encapsulation efficiency and in vitro drug release kinetics. The MTT assay depicted a high amount of cytotoxicity of GAR-NPs in B16F10, HepG2 and KB cells. A considerable amount of cell apoptosis was observed in B16f10 and KB cell lines. In vivo cellular uptake of fluorescent NPs on B16F10 cells was also investigated. Finally the GAR loaded NPs were radiolabeled with technetium-99m with >95% labeling efficiency and administered to B16F10 melanoma tumor bearing mice to investigate the in vivo deposition at the tumor site by biodistribution and scintigraphic imaging study. In vitro cellular uptake studies and biological evaluation confirm the efficacy of the formulation for cancer treatment.
Project description:The incidence of malignant melanoma is rising more rapidly than that of any other cancer in the United States. The melanocortin 1 receptor (MC1R) is overexpressed in most human melanoma metastases, thus making it a promising target for imaging and therapy of melanomas. We have previously reported the development of a peptidomimetic ligand with high specificity and affinity for MC1R. Here, we have conjugated near-infrared fluorescent dyes to the C-terminus of this ligand via lysine-mercaptopropionic acid linkers to generate MC1R specific optical probes (MC1RL-800, 0.4 nM K(i); and MC1RL-Cy5, 0.3 nM K(i)). Internalization of the imaging probe was studied in vitro by fluorescence microscopy using engineered A375/MC1R cells and B16F10 cells with endogenous MC1R expression. The in vivo tumor targeting of MC1RL-800 was evaluated by intravenous injection of probe into nude mice bearing bilateral subcutaneous A375 xenograft tumors with low MC1R expression and engineered A375/MC1R tumors with high receptor expression. Melanotic B16F10 xenografts were also studied. Fluorescence imaging showed that the agent has higher uptake values in tumors with high expression compared to low (p < 0.05), demonstrating the effect of expression levels on image contrast-to-noise. In addition, tumor uptake was significantly blocked by coinjection of excess NDP-?-MSH peptide (p < 0.05). In conclusion, the MC1R-specific imaging probe developed in this study displays excellent potential for the intraoperative detection of regional node involvement and for margin detection during melanoma metastasis resection.
Project description:Although targeted therapy and immunotherapy greatly improve the outcome of melanoma, drug resistance and low response rates still maintain the unsubstitutability of traditional chemotherapy. Cisplatin (CDDP) is widely used in different types of tumours with high response rates, but it generally has low efficiency in melanoma. The mechanisms underpinning the phenomena are not sufficiently understood. Here we demonstrated that various melanoma cell lines adopted senescence phenotype after CDDP treatment in contrast to the other types of tumour cells. CDDP treatment induced melanoma A375 cells into senescence through the sequential activation of the DNA damage response and the P53/P21 pathway. All the senescent melanoma cells induced by CDDP alone or the combination of CDDP and dacarbazine developed robust senescence-associated secretory phenotype (SASP), that is, the secretion of multiple cytokines. IL-1? was an early component and an upstream regulator of SASP. Similarly, CDDP either alone or combined with dacarbazine could induce melanoma cell senescence and SASP in either A375 or B16F10 melanoma xenograft mice. The supernatant of senescent A375 cells promoted the growth of normal non-senescent A375 cells and enhanced their expression and secretion of IL-8 through the activation of the ERK1/2-RSK1 pathway. The transplantation of non-senescent and senescent A375 cells together into nude mice showed accelerated tumour growth compared with transplanting non-senescent cells alone; no tumours developed when transplanting senescent cells alone. Following CDDP administration in A375-bearing mice, the intratumour injection of neutralisation antibodies targeting the SASP factors IL-1? or IL-8 evidently delayed tumour growth. The results suggest that the CDDP-induced senescent melanoma cells promote non-senescent cells proliferation through the activation of ERK1/2-RSK1 pathway by the SASP factors. Cell senescence and concomitant SASP may be the particular mechanisms for melanoma to resist chemotherapeutics.