| [Objective]The inflammation-triggered biosynthesis of LXA4is recognized as a transcellular process through cell-cell interactions. Two steps are required:(1) donor cell releases a metabolic intermediate of eicosanoid; and (2) vicinal acceptor cell takes up and converts the intermediate to LXA4. Neither donor cells nor acceptor cells can generate LXA4alone. Among the pathways of LXA4trans-cellular biosynthesis, leukocyte/platelet interaction is an important pattern. Two enzymes5-lipoxygenase (LO) and12-LO are in-volved in this process. In response to the stimulation of inflamma-tion, leukocytes utilize5-LO to catalyze arachidonic acid and release leukotriene A4. The latter is then taken up and converted to LXA4by12-LO in platelet. Another transcellular biosynthetic route identified is the sequential lipoxygenation of arachi-donic acid by15-LO in epithelial cells and monocytes and5-LO in neutrophils.Besides inflammatory signals, shear force can also activate and induce platelet apoptosis. As a result, activated or apopto-tic platelets may shed components of their plasma membranes encapsulating cytoplasmic elements into the extracellular space. These shed vesicles vary from100to1000nm in size and known as microparticles. Interestingly, microparticles seem to be capable of transferring cellular components from one cell to another. This leads us to assume that platelet-derived microparticles might transfer12-LO to leukotriene-producing [Methods]1. Isolation of plateletsWhole blood in3.8%sodium citrate was centrifuged for20min at250gto prepare platelet-rich plasma. Platelets were pelleted by centrifugation for20min at1500g. In some case, the pellet from250g centrifugation was used to isolate leukocytes.2. Isolation of microparticlesSupernatant from ADP-treated platelets or LPS-treated leukocytes were used to isolate microparticles. Briefly, supernatant was first centri-fuged for10min at2500g to get rid of cells and debris. Then the supernatant was further centrifuged for60min at14000gto pellet microparticles.3. Generation of bone marrow-derived mast cellsBone marrow cells from wild type or5-LO-/-mice (Jackson Lab-oratory) were cultured in vitro to generate mast cells according to previous description. Cells isolated from bone of Balb/c mice, and then lOng/ml IL-3and10ng/ml SCF were added. Cultured medium were half changed every three days and full cytokines were added. After3-4weeks induction, mast cells could be derived from bone marrow cells.4. Enzyme-linked immunoadsorbent assay (ELISA)To assess LXA4, the incubated supernatant or the peritoneal exudate was assayed by ELISA kit (Neogen, Lexington, KY). IL-10and IFN-y peritoneal exudate or IL-10, IFN-γ, TNF-α and IL-18in colon tissue were assessed by ELISA kits (R&D Systems).5. Real time RT-PCRFor real time RT-PCR assays, the cDNA sequences of genes were retrieved from NCBI database. The primers were designed with the Oligo Primer Analysis4.0software and the sequences were blasted (http://www.ncbi.nlm.nih.gov/BLAST/). Real time RT-PCR was done as described previously. The mRNA level of the detected gene was expressed as the relative level to that of GAPDH.6. Mouse colitis modelBALB/c mice (18-20g) were purchased from Center of Medical Experimental Animals of Hubei Province (Wuhan, China). Colitis was induced by the addition of DSS (5000Da, Sigma) to the drinking water. Groups of mice (n=6) were treated with3.5%DSS or regular water for the indicated days. Body weights were assessed every day during the treatment period. After sacrificed, the colon length of mice was measured by a technician blinded to the experimental conditions.[Results]1.12-LO is present in platelet-derived microparticlesTo address whether12-LO was present in platelet-derived microparticles, platelets were isolated from murine peripheral blood and incubated with or without ADP for2h, and then supernatants were used to prepare microparticles for Western blot analysis. Data suggested the presence of12-LO protein in platelet-derived microparticles.2. Mast cells take up platelet-derived microparticles and utilize12-LO to generate LXA4To verify whether mast cells took up12-LO containing platelet-derived microparticles and utilized12-LO to convert leukotriene A4to LXA4. We incubated murine bone marrow-derived mast cells, which contain presynthesized leukotriene A4, with platelet-derived microparticles for12h. The production of LXA4in the supernatant was detected; however, the supernatant from either mast cell or microparticle culture was absent of LXA4.3. LXA4production by mast cells taking up platelets-derived microparticles has anti-inflammatory effect in vivo We used LPS-triggered inflammation model to test the anti-inflammatory effect of platelets-derived microparticles in vivo.2μg LPS were i.p. injected to mice1h after microparticles injection. After another5h, the peritoneal inflammation was evaluated by the examination of a panel of pro-inflammatory and anti-inflammatory factors, including IFN-y, IL-6, IL-10, CCL2and iNOS. Data showed that IL-6, CCL2and iNOS were affected to a certain low extent, whereas IFN-y and IL-10were strikingly downregulated and upregulated, respectively.4. Mast cell-derived LXA4has inhibitory effects on DSS-induced inflammatory bowel diseaseWe further chose the DSS-induced mouse model of inflammatory bowel disease for the further functional study of microparticles-derived LXA4in vivo. The result showed that platelet-derived microparticles effectively prevented the weight loss, compared with control groups. Besides, the production of inflammatory mediators suchas IL-18, IFN-y and TNF-a were induced by DSS but were inhibited by platelet-derived microparticles or LXA4administration.[Conclusions] We show here that platelet-derived microparticles can transfer12-LO to mast cells, thus revealing a novel pathway for LXA4bio-synthesis in vivo. This newly discovered mechanism may not only fit the energy saving rule in living cells but also might act as the primary contributor of physiologic LXA4production. In a word, our data uncover a novel biological property of microparticles, which might open a new avenue in the study of inflammation and immune regulation. [Objective] A major challenge in cancer chemotherapy is the selective delivery of chemotherapeutic drugs to tumor cells. Recent studies have demonstrated that packaging clinically approved drugs into nanoscale vesicular vehicles (10-100nm in diameter) can effectively deliver chemotherapeutic drugs to tumor sites, leading to improved pharmacokinetic efficiency and therapeutic efficacy. Nevertheless, these drug-loaded artificial nanoparticles face several disadvantages. First, nanometer materials as nonself components, are usually toxic and may cause adverse effects, for example, by activating oxidative stress pathways; second, micelles are of inadequate stability in the blood stream; third, coencapsulation of more than one drug can result in batch-to-batch variability in the drug load; and fourth, the cost of assembly of nanoparticles is expensive, especially if they contain engineered antibodies or aptamers. Notwithstanding these defects, the development of advanced nanoparticle drug carriers is currently vigorously pursued in the field of cancer therapeutics. However, one facet or direction, often overlooked, is the existence and efficacy of natural counterparts to these artificial nanoparticles.Cells are capable of generating various vesicular vesicles of different sizes but whether these cell-derived microvesicles are suitable for drug delivery remains unclear. In response to various stimuli, cells may change their cytoskeletal structure and result in plasma membranes encapsulating cytosolic elements that are released into the extracellular space. These specialized subcellular vesicles are called microparticles (MPs) with100-1000nm in diameter. Wolf first observed MPs from activated blood platelets in1967and described them as pro-coagulant "dust". Since then, it has progressively become more evident that MPs can be derived from almost all cell types and play crucial roles under physiological or pathophysiological conditions. MPs not only contain messenger molecules, enzymes, RNAs and even DNA, but also are capable of transferring these bioactive molecules from one cell to another. Thus, functionally, MPs appear to act as vectors delivering molecular messages between cells. Considering the similarities of MPs and nanoparticles in size, structure and vector function, it is reasonable to hypothesize that MPs may be useful as endogenous natural vehicles to deliver chemotherapeutic drugs. This study demonstrates that MPs, derived from apoptotic tumor cells, can package and deliver chemotherapeutic drugs to tumor cells, thereby, leading to the timely death of the tumor cells without typical side effects.[Methods]1. Mice and cell lines.Female BALB/c and BALB/c-SCID mice,6to8-week-old, were purchased from Center of Medical Experimental Animals of Hubei Province (Wuhan, China) for studies approved by the Animal Care and Use Committee of Tongji Medical College. Murine hepatocarcinoma cell line H22and human ovarian cancer cell line A2780were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China), and cultured according to the guidelines given.2. Generation and isolation of MPs.Tumor cells were treated with chemotherapeutic drug methotrexate (MTX), hydroxyl camptothecin or cisplatin, and then exposed to ultraviolet irradiation (UVB,300J/m2) for1hour.12hours later, supernatants were used for MP isolation as described before. Briefly, supernatants were first centrifuged for10min at600g to get rid of cells and then centrifuged for2min at14000g to remove debris. At last the supernatant was further centrifuged for60min at14000g to pellet MPs. 3. MP Counting.A flow cytometry-based method was used to count the number of MPs. After centrifugation, the MPs were suspended with PBS that was pre-filtered through0.1μm filter and passed through1μm filter to further exclude background noise or non-specific events. The MPs mixed evenly with3μm latex beads (LB-30, Sigma, St Louis, MO) with a known number. For flow cytometric analysis,0.8μm deep blue dyed-latex beads (L1398, sigma) were first used for gating and voltage adjustment, since such beads are fluorescent and can be detected on FL4channel. When the mixture was analyzed by flow cytometry, each LB30bead formed a dot in the gate of the large size population.4. Transmission electron microscope.MPs were passed through1μm filter and fixed at room temperature for60minutes with4%paraformaldehyde in0.01M PBS. After washing with PBS, the preparations were postfixed in1%OsO4(Taab) for30minutes. After rinsing with distilled water, the pellets were dehydrated in graded ethanol, including block staining with1%uranylacetate in50%ethanol for30minutes, and embedded in Taab. After overnight polymerization at60℃and sectioning for EM, the ultrathin sections were analyzed with a JEM1010electron microscope (JEOL, Japan).5. Assay of MPs packaging chemotherapeutic agents.Doxorubicin, a red fluorescent chemotherapeutic drug, was used to treat H22or A2780tumor cells. The released MPs were isolated and observed under a two-photon fluorescent microscope (LSM710and ConfoCor3systems, Carl Zeiss, Germany) to confirm the formation of MPs packaging chemotherapeutic agents.6. High-performance liquid chromatography.The concentration of chemotherapeutic drug in MPs was measured by HPLC Briefly, H22tumor cell-derived, doxorubicin-packaging MPs were processed by lysis buffer, proteinase K, phenylmethylsulfonyl fluoride and DNase I according to previous description. The HPLC system consisted of a1525Binary HPLC Pump, a717Plus Autosampler and a2475Multi-Wavelength Fluorescence Detector (Waters Corporation, Milford, CT). Chromatography was performed on a column (4.6×150mm, particle size5μm). The effluents were monitored at an excitation wave length of480nm and an emission wave length of560nm at40℃. Detection and integration of chromatographic peaks was performed using Empower2software (Waters Corporation, Milford, CT).7. Cytotoxicity assay of drug-packaging MPs.Drug-packaging MPs were prepared and added to the cultured tumor cells. The cells were observed under a microscope at different time points. In some cases, cells were collected and stained with annexin V and propidium iodide for apoptosis detection by a flow cytometer.8. Labelling of MPs.Isolated MPs were labeled with a red-fluorescent cell linker (PKH26, Sigma, St Louis, MO) according to the manufacturer’s protocol. 9. Flow cytometry.A BDTM LSR II flow cytometer (BD) was used to do the flow cytometric analysis of the MPs in samples. The instrument settings and MP gating were adopted from previous works. Samples were diluted in1:30with1.2μm pore size membrane-filtered PBS in order to reduce background event numbers. A microbead of3μm in diameter (Sigma-Aldrich) was used to select optimal instrument settings and MP gate. Total event counts of MPs were determined within the MP gate.10. Animal model and MP treatment protocol.l×105H22murine hepatocarcinoma tumor cells (BALB/c background) were i.p. injected into BALB/c mice. MTX-packaging MPs were then administered to one mouse per packaged MPs. On day2after H22tumor cell inoculation, mice were received MTX-packaging MPs by i.p. injection once per day for5days.For the human ovarian cancer murine peritoneal model, BALB/c-SCID mice were i.p. injected with5×106A2780human ovarian cancer tumor cells. On day2after the inoculation, MPs were i.p. injected to mouse once per day for5days. After day14, an additional treatment was administered once per day for5days. In selected cases, drug-packaging MPs and paclitaxel-liposome were i.v. injected to mice.11. Hoechst33342uptake assay.Isolated MPs treated with or without0.5%Triton X-100were incubated with Hoechst33342for12hours. MPs were isolated again re-suspended in30μl PBS buffer. Suspensions were smeared on a glass slide and photographed under a fluorescence microscope.[Results]1. Development of tumor cell-derived drug-encapsulating MPs.We first tested whether apoptotic tumor cells, induced by chemotherapeutic drugs, could produce drug packaging MPs. For this purpose, CFSE-stained mouse hepatocarcinoma tumor cell line H22were incubated with10μg/ml methotrexate (MTX) and then irradiated with UV light for further apoptosis induction. The MPs were isolated, and analyzed by flow cytometry. Results showed that apoptotic tumor cells could indeed release considerable MPs2. Tumor cell-derived drug-encapsulating MPs are cytotoxic.To verify whether drug-encapsulating MPs are cytotoxic to tumor cells, low or high concentrations of MTX (1or10μg/ml) were added to the cultured H22cells (5×106)1hour before UV irradiation.12hours later, the released MTX-encapsulating MPs were incubated with H22cells. Both high and low MTX-MPs could induce tumor cell death after48or72hours.3. MTX-encapsulating MPs inhibit ascites hepatocarcinoma growth.We next investigated whether MPs could be used for anticancer therapy in vivo and injected MTX-encapsulating MPs i.v. or i.p. into mice.2mg MTX were added to2ml culture media with2×107H22tumor cells1hour before UV irradiation. Each of the prepared MPs was then used for one mouse per package. Collectively, mice were treated once per day for six days. Compared to simple MPs or PBS control, MTX-MPs did not induce any hair and/or weight changes in the mice or adversely affect liver or kidney functions. 4Cisplatin-encapsulating MPs inhibit human ovarian cancer growth. To further confirm that tumor cell derived chemotherapeutic drug-encapsulating MPs have an anticancer activity, human ovarian cancer in SCID mice was also tested by i.p. injection of A2780cells.600μg cisplatin were added to2ml culture media with2×107A2780cells1hour before UV irradiation. The MPs formed were then used for one mouse each. Similar to the above MTX-MPs, the administration of cisplatin-MPs continuously for five days did not affect liver and kidney functions in SCID mice either. Data also showed that A2780cell derived MPs inhibit human ovarian cancer growth by packaging cisplatin or cisplatin/paclitaxel.5. Tumor cells take up chemotherapeutic drug-packaging MPs.To elucidate the mechanism by which drug-encapsulating MPs efficiently mediate tumor cell killing, the first question asked was whether the interaction between MPs and tumor cells were required for tumor cell killing. Data suggested that MPs are efficiently taken up by tumor cells in vitro and in vivo.[Conclusions] The data in this study clearly show that tumor cell-derived MPs, by virtue of their biological formation and subsequent biochemical features, can be used as a carrier to deliver therapeutic agents to tumor cells, leading to effective tumor cell killing with reduced adverse effects. This study may open a new aspect of MP biology and may have discovered a new way for chemotherapeutic drug delivery in cancer therapy. |
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