Gheibi, Dr. Nematollah

pourfarzad, farnaz

Qazvin University of Medical Sciences Repository

Liposome encapsulated luteolin showed enhancedantitumor efficacy to colorectal carcinomaPresented by : farnaz pourfarzadSupervisor: Dr. nematollah qeibiMedical biotechnology groupFaculty of paramedicineQazvin university of medical sience123Research trend Last update:3/10/20214introduction•Colorectal carcinoma ( CRC ) :• third most common form of cancer• rectum exhibits common internal malignancies (1).• Treatment options :• radiotherapy, chemotherapy, surgery• outcomes are limited           cancer recurrence, toxicity on the human body, neurotoxicity, gastrointestinal reaction, kidney failure, and cardiotoxicity (2,3).• Chemoprevention           to reverse, suppress, or prevent molecular or histologic premalignant lesions from progressing to invasive cancer by using natural or synthetic agents (4).5introduction•CRC           rationale cancer for chemoprevention studies, due to high incidence of pre-neoplastic lesions and cancerous tumors (5).• ideal chemopreventive compound          potent, highly effective, less expensive, easily available (3).• Luteolin (3',4',5,7-tetrahydroxyflavone) flavonoid poly-phenolic compound found in many plant types• Biological activities           anti-inflammatory, anti-allergy, anti-cancer against lung, head and neck, prostate, breast, colon, skin cancers via  inducing apoptosis, suppressing metastasis, and angiogenesis (6,7,8). 6introduction• Luteolin           poor aqueous solubility• low bioavailability after oral administration• difficult to make intravenous or intraperitoneal administration• Liposomes:• longer blood circulation time • higher biocompatibility and excellent bioavailability• higher tumor-specific delivery (9,10,11). 7Materials and methods8Liposome preparation• thin film hydration method :1. mixtures of luteolin/cholesterol/lecithin in 1:2:7 weight ratios2. dissolved in ethanol, were transferred into a round bottom flask3. The flask connected to a rotary evaporator at 50 rpm and water bath at 40˚C4. Vacuum evaporate the ethanol, form a homogeneous lipid film on the flask wall5. the lipid film hydrated in normal saline by rotating the flask at 37˚C6. luteolin liposome was sonicated with 50 watts of power for 10 min9Size distribution and ζ-potential•mean particle sizes and ζ-potential:• photon correlation spectroscopy using Malvern Zeta sizer 3000 HS at 25˚C• form feature of the liposomes:• transmission electron microscope (TEM)• negative staining method with 1% sodium phosphotungstate solution for 2 min at25˚C10Drug loading and encapsulation efficiency1. prepared liposomes were solubilized in methanol2. cyclomixer extract the drug from lipid to methanol3. HPLC           octadecylsilyl column, mixture of acetonitrile and 0.1% formic acid as mobile phase, flow rate at 0.5 ml/min4. UV detection wavelength at 350 nm• soluble unencapsulated drug was measured by ultrafiltration using centrifugal filter tubes with a molecular weight cut-off of 300 kDa• Drug loading and encapsulation efficiency:11Drug release• dialysis method : for in vitro1. Lipo-Lut and Free-Lut in dimethyl sulphoxide (DMSO) solution placed in a dialysis bag2. dialysis bag placed in a 50ml PBS supplemented with 0.5% Tween-203. shaking incubator with stirring speed of 100 rpm at 37˚C4. The amount of luteolin released at each time-point was determined using HPLC• in vitro:1. BALB/c mice, treated with Free-Lut or Lipo-Lut, dose of 50 mg/kg body weight via IVinjection2. Blood collected, centrifuged at 8,000 rpm for 10 min, diphenhydramine added,3. drug extracted from plasma samples, HPLC analysis to determine luteolin levels12MTT assay• CT26 colorectal carcinoma cell line, RPMI-1640, 10% fetal bovine serum, 5% CO2 incubator at 37˚C• Cells at logarithmic growth phase placed in a 96-well plate at 37˚C overnight• treated with various concentrations of Free-Lut or Lipo-Lut and cultured for 24, additional 3 h of culture with 0.5 mg/ml MTT at 37˚C• samples analyzed using a microplate reader13Assessment of apoptosis• flow cytometry  Annexin V-fluorescein sothiocyanate (FITC) and propidiumiodide (PI)1. Treated cells incubated for 24 h at 37˚C in 6-well plates2. cells were trypsinized, washed and resuspended in PBS3. treated with Annexin V-FITC and PI in the dark for 15 min.4. Flow cytometry analysis was performed with an EpicsXL Coulter flow cytometer 14Mouse tumor model• Female BALB/c mice, 6 weeks old and weighting 18-20 g• The murine tumor models established by subcutaneous inoculation in the right flanks of female BALB/c mice with CT26 cells• The drug treatments were initiated when tumors reached the volume of 100 mm3, day 7 post-cell inoculation, dose of 50 mg/kg• Groups of normal saline (NS), empty liposome (EM-Lipo), Free-Lut, and Lipo-Lut via the tail vein every 2 days a total of five times• the mice were sacrificed on the day 22, and the tumors were excised, weighed and fixed in 10% neutral buffered formalin solution or frozen at -80˚C 15Hematoxylin and eosin (H&E) staining and immunofluorescence staining• CT26 xenograft fixed in 4% paraformaldehyde for 12 h and embedded in paraffin• Sections (5-µm thick) were cut, dewaxed, rehydrated, and stained with H&E• inhibitory effect on neovascularization frozen tissues sectioned (5 µm), fixed in acetone• tissues sections incubated with monoclonal anti-CD31 antibody, flowing stained with a secondary goat antibody (FITC) , number of microvessels counted• immunofluorescence of Ki-67 expression monoclonal anti-Ki67 antibody, FITC labelled goat anti-mouse secondary antibody • apoptotic cells detected with TUNEL Detection kit 16Statistical analysis• SPSS statistics 17.0• Quantitative data expressed as the mean ± standard deviation of three independent experiments and analyzed by one-way ANOVA• Comparison between the groups was made by analyzing data with S-N-K method• A P-value <0.05 was considered to indicate a statistically significant difference17Results18Characterization of Lipo-Lut• dynamic light scattering results:• diameter of Lipo-Lut was around 105 nm• ζ-potential of Lipo-Lut was 0.12 mv• TEM results :• Lipo-Lut was spherical and had a regular shape• Free-Lut stratified in water, precipitated in the bottom of the bottle• Lipo-Lut can be stably suspended in water solution• drug loading and encapsulation efficiency 10 and 90%, for the Lipo-Lut formulation 19Characterization of Lipo-Lut20Drug release and Pharmacokinetics of Lipo-Lut• luteolin released slowly from the liposomes, Free-Lut released very quickly• cumulative percentage release           the amount of drugreleased from liposomes gradually increasedover time, after 120 h increased over 80%• free drug exhibited high level (80%) at 8 h21• the liposome improved bioavailability of poor water soluble drugs         mean plasma concentration-time profiles of luteolin after IV administration of free and liposome drug• Free-Lut rapidly cleared, plasma levels were lessthan 50% of the injected dose within 5 min of injection• luteolin concentration in plasma was almost 10-fold higher for Lipo-Lut at 2 h after drug injection• liposomal encapsulation reduced drug elimination   Drug release and Pharmacokinetics of Lipo-Lut22Tumoricidal effects on colorectal cancer cells• Lipo-Lut exhibited inhibitory effect, as similar to Free-Lut, both drugs showed dose-dependent inhibition of cell growth• After 24h of incubation, Lipo-Lut showed higherinhibition compared to the Free-Lut atall the concentrations• empty liposomes did not show any toxicity• Lipo-Lut inhibited tumor proliferation in vitro 23• the inhibitory effect of Lipo-Lut involved the initiation of apoptosis• flow cytometry analysis of Annexin V staining for phosphatidylserine• CT26 cells were divided into four groups:• necrotic cells (upper left quadrant, Annexin V-/PI+)• healthy viable cells (lower left quadrant, Annexin V-/PI-)• cells in the early apoptosis stage (lower right quadrant, Annexin V+/PI-)• cells that are in late apoptosis or already dead (upper right quadrant, Annexin V+/PI+)• more than 95% of control CT26 cells were viable, all the cells incubated with Lipo-Lut or Free-Lut displayed evidence of apoptosisTumoricidal effects on colorectal cancer cells24Tumoricidal effects on colorectal cancer cells25Tumoricidal effects on the mouse tumor model• CT26 colorectal carcinoma graft model in BALB/c mice, 22-days of treatment• Four groups of  NS, EM-Lipo, Free-Lut, Lipo-Lut• the final tumor volume of mice treated with luteolin was notably reduced• Lipo-Lut group exhibited the most significant inhibitory efficacy• no significant difference between NS group and EM-Lipo group• The smallest tumor weights were observed in the Lipo-Lut group• the treatment of Lipo-Lut resulted in a robust efficacy in reducing tumor volume 26Tumoricidal effects on the mouse tumor model27Lipo-Lut induced apoptosis• TUNEL assay• a significant number of apoptotic cells appeared with green fluorescence in the tumor tissue of luteolin-treated mice • Treatment with Lipo-Lut produced more apoptotic cells than treatment with the Free-Lut28Lipo-Lut inhibited tumor vascularization• immunofluorescence analysis with anti-CD31 monoclonal antibody• CD31-positive endothelial cells in luteolin treated groups had weaker fluorescence than those of NS group and EM-Lipo group• less number of microvessels in the Free-Lut group and Lipo-Lut group compared to the control group (P<0.01)• Lipo-Lut group reduced number of microvessels more remarkably compared with Free-Lutgroups (P<0.01)• No significant difference was observed between NS group and EM-Lipo group (P>0.05)29Lipo-Lut inhibited tumor vascularization30Lipo-Lut decreased Ki-67 expression• The expression of Ki-67 is associated with tumor cell proliferation and growth,widely used in routine pathologicalinvestigation as a proliferation marker • Immunofluorescence examinationof Ki-67 staining revealed an inhibitionof proliferating cells in the Lipo-Lut-treated mice31Discussion• liposomes          safe, well tolerated and potential drug delivery system • best-known formulation is liposome-encapsulated doxorubicin (Doxil or Caelyx) (12,13).• less cardiotoxicity, providing comparable antitumor activity (14,15).• the actions of some poor water soluble drugs enhanced after encapsulation into liposome• normal endothelium gaps         5-10 nm• tumor capillaries gaps          100 to 780 nm (16)• Water soluble lipo-lut size           ~105 nm• Lipo-Lut could prolong the drug release, bioavailability of Lipo-Lut improved• Lipo-Lut have more tumor growth inhibition activities than Free-Lut on CT26 cells 32• the antitumor activities of Lipo-Lut on CT26 cells grafts in BALB/c mice• stronger tumor growth-inhibiting effects• suppressed angiogenesis, increased apoptosis• Angiogenesis plays a important role in tumor growth and invasion, The most essential factor is vascular endothelial growth factor (VEGF) • luteolin could inhibit VEGF-stimulated endothelial cell proliferation, migration, invasion and tumor angiogenesis• A study from Norhaizan           luteolin induced apoptosis in colon cancer by modulating the expressions of bax, Bcl-2 and caspase 3 in vitro and in vivo• luteolin acted against DNA damage and activated DNA repair mechanism in Caco-2 colon cancer cells (17,18).          Discussion33• accumulation of liposome in the tumor microenvironment is more than normal tissues (19,20)• leaky vasculature of the tumor tissue           provide a channel allowing liposome to more easily target tumor tissue (1).• solid tumors usually lack effective lymphatic drainage• The Lipo-Lut inhibited activity of tumor growth more effectively than the Free-Lut in both CT26 cells and mouse tumor model of colorectal carcinoma• Mechanism of action :• Induction of apoptosis in tumor cells • reduced tumor angiogenesis, blocking the nutrition supply into tumor tissue• Lipo-Lut inhibited tumor proliferation Discussion34References1. . Yang C, Liu HZ, Fu ZX and Lu WD: Oxaliplatin long-circulating liposomes improved therapeutic index of colorectal carcinoma. BMC Biotechnol 11: 21, 2011.2. . Chen AY and Chen YC: A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention. Food Chem 138: 2099-2107, 2013.3. Majumdar D, Jung KH, Zhang H, Nannapaneni S, Wang X, Amin AR, Chen Z, Chen ZG and Shin DM: Luteolin nanoparticle in chemoprevention: In vitro and in vivo anticancer activity. Cancer Prev Res (Phila) 7: 65-73, 2014.4. Madhunapantula SV and Robertson GP: Chemoprevention of melanoma. Adv Pharmacol 65: 361-398, 2012.5. . Benbrook DM, Guruswamy S, Wang Y, Sun Z, Mohammed A, Zhang Y, Li Q and Rao CV: Chemoprevention of colon and small intestinal tumorigenesis in APC(min/+) mice by SHetA2 (NSC721689) without toxicity. Cancer Prev Res (Phila) 6: 908-916, 2013.6. Yang MY, Wang CJ, Chen NF, Ho WH, Lu FJ and Tseng TH: Luteolin enhances paclitaxel-induced apoptosis in human breast cancer MDA-MB-231 cells by blocking STAT3. Chem Biol Interact 213: 60-68, 2014.7. Huang X, Dai S, Dai J, Xiao Y, Bai Y, Chen B and Zhou M: Luteolin decreases invasiveness, deactivates STAT3 signaling and reverses interleukin-6 induced epithelial-mesenchymal transition and matrix metalloproteinase secretion of pancreatic cancer cells. Onco Targets Ther 8: 2989-3001, 2015.8. Yan M, Liu Z, Yang H, Li C, Chen H, Liu Y, Zhao M and Zhu Y: Luteolin decreases the UVA-induced autophagy of human skin fibroblasts by scavenging ROS. Mol Med Rep 14: 1986-1992, 20169. Yuan Y, Zhao Y, Xin S, Wu N, Wen J, Li S, Chen L, Wei Y, Yang H and Lin S: A novel PEGylated liposome-encapsulated SANT75 suppresses tumor growth through inhibiting hedgehog signaling pathway. PLoS One 8: e60266, 2013 3510. . Fetterly GJ, Grasela TH, Sherman JW, Dul JL, Grahn A, Lecomte D, Fiedler-Kelly J, Damjanov N, Fishman M and Kane MP: Pharmacokinetic/pharmacodynamic modeling and simulation of neutropenia during phase I development of liposome-entrapped paclitaxel. Clin Cancer Res 14: 5856-5863, 2008.11. Gabizon A, Catane R, Uziely B, Kaufman B, Safra T, Cohen R, Martin F, Huang A and Barenholz Y: Prolonged circulation time and enhanced accumulation in malignant exudates of doxo- rubicin encapsulated in polyethylene-glycol coated liposomes. Cancer Res 54: 987-992, 1994.12. Barenholz Y: Doxil® - the first FDA-approved nano-drug: Lessons learned. J Control Release 160: 117-134, 2012.Schiffelers RM, Metselaar JM, Fens MH, Janssen AP, Molema G and Storm G: Liposome-encapsulated prednisolone phosphate inhibits growth of established tumors in mice. Neoplasia 7: 118-127, 2005.13. HarrisL, Batist G, BeltR, RoviraD, NavariR, AzarniaN, WellesLand Winer E; TLC D-99 Study Group: Liposome-encapsulated doxorubicin compared with conventional doxorubicin in a randomized multicenter trial as first-line therapy of metastatic breast carcinoma. Cancer 94: 25-36, 2002.14. . Hioki A, Wakasugi A, Kawano K, Hattori Y and Maitani Y: Development of an in vitro drug release assay of PEGylated liposome using bovine serum albumin and high temperature. BiolPharm Bull 33: 1466-1470, 2010.15. Deshpande PP, Biswas S and Torchilin VP: Current trends in the use of liposomes for tumor targeting. Nanomedicine (Lond) 8: 1509-1528, 2013.16. Haley B and Frenkel E: Nanoparticles for drug delivery in cancer treatment. Urol Oncol 26: 57-64, 2008.36References17. Pandurangan AK and Esa NM: Luteolin, a bioflavonoid inhibits colorectal cancer through modulation of multiple signaling pathways: A review. Asian Pac J Cancer Prev 15: 5501-5508, 201418. Wang LM, Xie KP, Huo HN, Shang F, Zou W and Xie MJ: Luteolin inhibits proliferation induced by IGF-1 pathway dependent ERα in human breast cancer MCF-7 cells. Asian Pac J Cancer Prev 13: 1431-1437, 2012.19. . Matsumura Y and Maeda H: A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropicaccumulation of proteins and the antitumor agent smancs. Cancer Res 46: 6387-6392, 1986.20. Vasey PA, Kaye SB, Morrison R, Twelves C, Wilson P, Duncan R, Thomson AH, Murray LS, Hilditch TE, Murray T, et al: Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: First member of a new class of chemotherapeutic agents-drug-polymer conjugates. Cancer research campaign phase I/II committee. Clin Cancer Res 5: 83-94, 1999.37References

Liposome encapsulated luteolin showed enhanced antitumor efficacy to colorectal (ارائه ژورنال کلاب)

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Liposome encapsulated luteolin showed enhanced antitumor efficacy to colorectal (ارائه ژورنال کلاب)

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