Search JIM Advanced Search

Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (1): 20-34.doi: 10.1016/S2095-4964(14)60003-0

• Research Article • Previous Articles     Next Articles

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

Li-na Wanga,b,c, Yuan Wanga,b,c,d, Yuan Lue, Zi-fei Yina, Yuan-hui Zhanga,b,c,d, George V. Aslanidib,c, Arun Srivastavab,c,f,g,h, Chang-quan Linga,d, Chen Lingb,c   

  1. Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai 200433, China
    Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    Department of Orthopaedics and Rehabilitation, University of Florida College of Medicine, Gainesville,Florida 32611, USA
    Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Genetics Institute, University of Florida College of Medicine, Gainesville, Florida 32611, USA
    Shands Cancer Center, University of Florida College of Medicine, Gainesville, Florida 32611, USA
  • Received:2013-07-22 Accepted:2013-08-27 Online:2014-01-10 Published:2014-01-15


In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.

Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. 


We observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. Conclusion

These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Key words: Celastrol, Pristimerin, Adeno-associated viral vector, Proteosomal inhibitor, Gene therapy

Figure 1

Schematic outline of chemical compounds and viral genomesA: The chemical structure of celastrol and pristimerin. B: Schematic structures of rAAV2 vectors containing various indicated rAAV vector genomes. sc: self-complementary; ss: single-stranded; AAV: adeno-associated virus; CBAp: chicken β-actin/cytomegalovirus enhancer hybrid promoter; EGFP: enhanced green fluorescence protein; EYFP: enhanced yellow fluorescence protein; HP: hairpin structure; HP-: hairpin structure without terminal resolution site; hGH: human growth hormone."

Figure 2

Celastrol modestly enhanced rAAV2-mediated transduction efficiency HeLa cells were treated with celastrol for 2 h, followed by infection with scAAV2-CB-EGFP vectors at multiplicity of infection of 5 000 viral genomes/cell with or without celastrol. All transgene expression was detected by fluorescence microscopy 72 h post-transduction and was analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel2) per visual field. Data are expressed as mean ± standard deviation, n= 3; **P<0.01, vs DMSO group.rAAV: recombinant adeno-associated virus; PBS: phosphate-buffered saline; DMSO: dimethyl sulfoxide."

Figure 3

Cell viability assays to determine IC50 of celastrol and pristimerin HeLa cells were exposed to different concentrations of celastrol or pristimerin for 4 h and then were cultured in complete Dulbecco’s modified Eagle medium without drugs for additional 20 h, followed by cell cytotoxicity assays using Cell Counting Kit-8.IC50: half maximal inhibitory concentration."

Figure 4

Pristimerin significantly enhanced rAAV2-mediated transgene expression in vitro A: Various doses of celastrol (1, 2.5 and 5 μmol/L) and pristimerin (0.5, 1 and 2.5 μmol/L) were co-administered with scAAV2-CBAp-EGFP (5 000 viral genomes/cell) vector to transduce HeLa cells. B: Transgene expression was detected by flow cytometry 72 h post-transduction from Figure 4A. C: Various doses of celastrol and pristimerin, as Figure 4A, were co-administered with high dose of scAAV2-CBAp-EGFP (50 000 viral genomes/cell) vector to transduce HeLa cells. D: Huh7 cells were treated with indicated drugs for 2 h, followed by infection with scAAV2-CBAp-EGFP vectors at 20 000 viral genomes/cell with or without drug treatment. All transgene expression was detected by fluorescence microscopy 72 h post-transduction and was analyzed quantitatively by ImageJ analysis software. Transgene expression was assessed as total area of green fluorescence (pixel2) per visual field. Data are expressed as mean ± standard deviation, n=3; **P<0.01, vs DMSO group; △△P<0.01, vs celastrol (1 μmol/L) group.rAAV: recombinant adeno-associated virus; DMSO: dimethyl sulfoxide. scAAV2-CBAp-EGFP: self-complementary adeno-associated virus 2-chicken β-actin/cytomegalovirus enhancer hybrid promoter-enhanced green fluorescence protein."

Figure 5

Total RNAs from HeLa cells were extracted 24 h post-infection with scAAV2 vectors RNAs were subjected to reverse transcription using oligo-d(T) primers, and subsequent qRT-PCR assays specific for enhanced green fluorescence protein. Data are expressed as mean ± standard deviation, n=3; *P<0.05, **P<0.01, vs DMSO group scAAV: self-complementary adeno-associated virus; qRT-PCR: quantitativereverse transcriptase-polymerase chain reaction; DMSO: dimethyl sulfoxide."

Figure 6

Pristimerin acted at a step post-viral entry A: Western blot assays showing the purity of isolated cellular fraction. B: Normalized percentages of the distributions of scAAV2 vectors in the nuclear fractions at 16 h post-treatment of indicated drugs with scAAV2 vectors. C: Numbers of intracellular scAAV2 vector genomes at 2 h post-treatment of indicated drugs with scAAV2 vectors. Data are expressed as mean ± standard deviation, n=3; **P<0.01, vs DMSO group.scAAV: self-complementary adeno-associated virus; DMSO: dimethyl sulfoxide."

Figure 7

Subcellular localization of scAAV2-Cy3 virions 16 h after treatment of indicated drugs Images were acquired on Leica TCS SP5 confocal microscopy using oil immersed 63× objective lens.Green: nucleus; Blue: cell membrane; Red: rAAV2 particles.scAAV: self-complementary adeno-associated virus; rAAV: recombinant adeno-associated virus."

Figure 8

Accumulation of poly-ubiquitinated total cellular proteins after pristimerin treatment HeLa cells were treated with indicated drugs for 2 h, followed by infection with scAAV2-CBAp-EGFP vectors at 2 000 viral genomes/cell with or without drug treatment for 2 h. Total cellular proteins were extracted, followed by Western blot assay using specific antibodies to ubiquitin.GAPDH is served as a loading control. GAPDH: glyceraldehyde 3-phosphate dehydrogenase."

Figure 9

Pristimerin enhanced rAAV2-mediated transgene expression in vivo without change of viral tropism Six- to eight-week old male immune-deficient NSG mice were treated for 3 d with pristimerin or celastrol at a dose of 4 mg/(kg·d) or a DMSO vehicle control by intraperitoneal administration. ssAAV2-CBAp-FLuc-EYFP vectors were systemically delivered via tail vein at 1×1011 viral genomes/mouse on the second day of treatment. A: Representative images from luciferase live imaging at 2-week and 8-week post-transduction. B and C: Major organs were harvested 8 weeks after transduction and sections of major tissues were examined for EYFP expression, using a fluorescence microscope. Representative images are shown. Original magnification, B ×50 and C ×50 or ×400. D: The quantitative data of fluorescence in mice liver. E: Vector genome copy numbers in liver 8 weeks after transduction. Total DNA was isolated from 25 mg tissues and 100 ng of each was used to determine vector genome copies. Data are expressed as mean ± standard deviation, n=3; **P<0.01, vs DMSO group.ssAAV: single-stranded adeno-associated virus; rAAV: recombinant adeno-associated virus; DMSO: dimethyl sulfoxide."

Figure 10

Transduction efficiency in vivo mediated by capsid-modified ssAAV2 after pristimerin treatmentA: Representative images from luciferase live imaging at 4-week post-transduction with indicated ssAAV2 vectors and cotreatment with indicated drugs. B: Quantification data of the luciferase expression over time. Data are expressed as mean ± standard deviation, n=3; △△P<0.01, vs rAAV2-TM group; ▲▲P<0.01, vs rAAV2-WT group.ssAAV: single-stranded adeno-associated virus; rAAV: recombinant adeno-associated virus; DMSO: dimethyl sulfoxide."

Figure 11

Transduction efficiency in vivo mediated by oversized wild-type scAAV8 vectors after pristimerin treatment A: Representative images from luciferase live imaging at 4-week post-transduction with oversized scAAV8 vectors and cotreatment with indicated drugs. B: Quantification data of luciferase expression in A. Data are expressed as mean ± standard deviation, n=3; □□P<0.01, vs rAAV8 group.scAAV: self-complementary adeno-associated virus; rAAV: recombinant adeno-associated virus; DMSO: dimethyl sulfoxide."

[1] Atchison RW, Casto BC, Hammon WM . Adenovirus-associated defective virus particles[J]. Science, 1965,149(3685):754-756
doi: 10.1126/science.149.3685.754 pmid: 14325163
[2] Lebkowski JS , McNally MM, Okarma TB, Lerch LB.Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types[J]. Mol Cell Biol, 1988,8(10):3988-3996
doi: 10.1128/MCB.8.10.3988
[3] Mingozzi F, High KA . Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges[J]. Nat Rev Genet, 2011,12(5):341-355
doi: 10.1038/nrg2988 pmid: 21499295
[4] Daya S, Berns KI . Gene therapy using adeno-associated virus vectors[J]. Clin Microbiol Rev, 2008,21(4):583-593
doi: 10.1128/CMR.00008-08
[5] Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, Viswanathan A, Holder GE, Stockman A, Tyler N, Petersen-Jones S, Bhattacharya SS, Thrasher AJ, Fitzke FW, Carter BJ, Rubin GS, Moore AT, Ali RR . Effect of gene therapy on visual function in Leber’s congenital amaurosis[J]. N Engl J Med, 2008,358(21):2231-2239
doi: 10.1056/NEJMoa0802268 pmid: 18441371
[6] Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J , Dell’Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J.Safety and efficacy of gene transfer for Leber’s congenital amaurosis[J]. N Engl J Med, 2008,358(21):2240-2248
doi: 10.1056/NEJMoa0802315 pmid: 18441370
[7] Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM, Flotte TR, Fishman GA, Heon E, Stone EM, Byrne BJ, Jacobson SG, Hauswirth WW . Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics[J]. Proc Natl Acad Sci U S A, 2008,105(39):15112-15117
doi: 10.1073/pnas.0807027105
[8] Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C , McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O’Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM.Adenovirus-associated virus vector-mediated gene transfer in hemophilia B[J]. N Engl J Med, 2011,365(25):2357-2365
doi: 10.1056/NEJMoa1108046 pmid: 22149959
[9] Hwu WL, Muramatsu S, Tseng SH, Tzen KY, Lee NC, Chien YH, Snyder RO, Byrne BJ, Tai CH , Wu RM.Gene therapy for aromatic L-amino acid decarboxylase deficiency[J].Sci Transl Med, 2012, 4(134):134ra61
doi: 10.1126/scitranslmed.3003640 pmid: 22593174
[10] Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM . Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy[J]. Proc Natl Acad Sci U S A, 2002,99(18):11854-11859
doi: 10.1073/pnas.182412299
[11] McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ . Adeno-associated virus terminal repeat(TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo[J]. Gene Ther, 2003,10(26):2112-2118
doi: 10.1038/
[12] Wang Z, Ma HI, Li J, Sun L, Zhang J, Xiao X . Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo[J]. Gene Ther, 2003,10(26):2105-2111
doi: 10.1038/
[13] Nathwani AC, Gray JT, Ng CY, Zhou J, Spence Y, Waddington SN, Tuddenham EG, Kemball-Cook G , McIntosh J, Boon-Spijker M, Mertens K, Davidoff AM.Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver[J]. Blood, 2006,107(7):2653-2661
doi: 10.1182/blood-2005-10-4035
[14] Wang X, Zhang A, Sun H, Wang P . Systems biology technologies enable personalized traditional Chinese medicine: a systematic review[J]. Am J Chin Med, 2012,40(6):1109-1122
doi: 10.1142/S0192415X12500826
[15] Zheng WL, Ma WL . Gene therapy through digestive tract — the elucidation of the mechanism of traditional Chinese medicine[J].Ke Ji Dao Bao, 1994(12):8-11
[16] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ . Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin[J]. Onkologie, 2006,29(8-9):367-371
[17] Zhang FL, Jia SQ, Zheng SP, Ding W . Celastrol enhances AAV1-mediated gene expression in mice adipose tissues[J]. Gene Ther, 2011,18(2):128-134
doi: 10.1038/gt.2010.120 pmid: 20844567
[18] Mitchell AM, Li C, Samulski RJ . Arsenic trioxide stabilizes accumulations of adeno-associated virus virions at the perinuclear region, increasing transduction in vitro and in vivo[J]. J Virol, 2013,87(8):4571-4583
doi: 10.1128/JVI.03443-12
[19] Duan D, Yue Y, Yan Z, Yang J, Engelhardt JF . Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus[J]. J Clin Invest, 2000,105(11):1573-1587
doi: 10.1172/JCI8317
[20] Douar AM, Poulard K, Stockholm D, Danos O . Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation[J]. J Virol, 2001,75(4):1824-1833
doi: 10.1128/JVI.75.4.1824-1833.2001
[21] Ding W, Zhang LN, Yeaman C , Engelhardt JF.rAAV2 traffics through both the late and the recycling endosomes in a dose-dependent fashion[J]. Mol Ther, 2006,13(4):671-682
doi: 10.1016/j.ymthe.2005.12.002 pmid: 16442847
[22] Buffa Filho W, Corsino J , Bolzani da SV, Furlan M, Pereira AM, Fran®a SC.Quantitative determination for cytotoxic Friedo-nor-oleanane derivatives from five morphological types of Maytenus ilicifolia(Celastraceae) by reverse-phase high-performance liquid chromatography[J]. Phytochem Anal, 2002,13(2):75-78
doi: 10.1002/pca.v13:2
[23] Brinker AM, Ma J, Lipsky PE, Raskin I . Medicinal chemistry and pharmacology of genus Tripterygium(Celastraceae)[J]. Phytochemistry, 2007,68(6):732-766
doi: 10.1002/chin.200727218 pmid: 17250858
[24] Yang H, Liu J, Dou QP . Targeting tumor proteasome with traditional Chinese medicine[J]. Curr Drug Discov Technol, 2010,7(1):46-53
doi: 10.2174/157016310791162785
[25] Yang H, Chen D, Cui QC, Yuan X, Dou QP . Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice[J]. Cancer Res, 2006,66(9):4758-4765
doi: 10.1158/0008-5472.CAN-05-4529
[26] Yang H, Landis-Piwowar KR, Lu D, Yuan P, Li L, Reddy GP, Yuan X, Dou QP . Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells[J]. J Cell Biochem, 2008,103(1):234-244
doi: 10.1002/jcb.21399 pmid: 17541980
[27] Wu J, Zhao W, Zhong L, Han Z, Li B, Ma W, Weigel-Kelley KA, Warrington KH, Srivastava A . Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity[J]. Hum Gene Ther, 2007,18(2):171-182
doi: 10.1089/hum.2006.088
[28] Wang Y, Ling C, Song L, Wang L, Aslanidi GV, Tan M, Ling C, Srivastava A . Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation[J]. Hum Gene Ther Methods, 2012,23(4):225-233
doi: 10.1089/hgtb.2012.090
[29] Ling C, Lu Y, Kalsi JK, Jayandharan GR, Li B, Ma W, Cheng B, Gee SW , McGoogan KE, Govindasamy L, Zhong L, Agbandje-McKenna M, Srivastava A.Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3[J]. Hum Gene Ther, 2010,21(12):1741-1747
doi: 10.1089/hum.2010.075
[30] Ling C, Lu Y, Cheng B, McGoogan KE, Gee SW, Ma W, Li B, Aslanidi GV , Srivastava A.High-efficiency transduction of liver cancer cells by recombinant adeno-associated virus serotype 3 vectors[J].J Vis Exp, 2011( 49):pii: 2538
[31] Song L, Kauss MA, Kopin E, Chandra M, Ul-Hasan T, Miller E, Jayandharan GR, Rivers AE, Aslanidi GV, Ling C, Li B, Ma W, Li X, Andino LM, Zhong L, Tarantal AF, Yoder MC, Wong KK Jr, Tan M, Chatterjee S, Srivastava A . Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo[J]. Cytotherapy, 2013,15(8):986-998
doi: 10.1016/j.jcyt.2013.04.003
[32] Ding W, Yan Z, Zak R, Saavedra M, Rodman DM, Engelhardt JF . Second-strand genome conversion of adeno-associated virus type 2(AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia[J]. J Virol, 2003,77(13):7361-7366
doi: 10.1128/JVI.77.13.7361-7366.2003
[33] Glickman MH, Ciechanover A . The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[J]. Physiol Rev, 2002,82(2):373-428
doi: 10.1152/physrev.00027.2001
[34] Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL, Handgretinger R . Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells[J]. J Immunol, 2005,174(10):6477-6489
doi: 10.4049/jimmunol.174.10.6477
[35] Markusic DM, Herzog RW, Aslanidi GV, Hoffman BE, Li B, Li M, Jayandharan GR, Ling C, Zolotukhin I, Ma W, Zolotukhin S, Srivastava A, Zhong L . High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines[J]. Mol Ther, 2010,18(12):2048-2056
doi: 10.1038/mt.2010.172
[36] Zincarelli C, Soltys S, Rengo G, Rabinowitz JE . Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection[J]. Mol Ther, 2008,16(6):1073-1080
doi: 10.1038/mt.2008.76
[37] Monahan PE, Lothrop CD, Sun J, Hirsch ML, Kafri T, Kantor B, Sarkar R, Tillson DM, Elia JR, Samulski RJ . Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application[J]. Mol Ther, 2010,18(11):1907-1916
doi: 10.1038/mt.2010.170
[38] Pajusola K, Gruchala M, Joch H, Lüscher TF, Yl®-Herttuala S, Büeler H . Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells[J]. J Virol, 2002,76(22):11530-11540
doi: 10.1128/JVI.76.22.11530-11540.2002
[39] Yan Z, Zak R, Zhang Y, Ding W, Godwin S, Munson K, Peluso R, Engelhardt JF . Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia[J]. J Virol, 2004,78(6):2863-2874
doi: 10.1128/JVI.78.6.2863-2874.2004
[40] Luo DQ, Wang H, Tian X, Shao HJ, Liu JK . Antifungal properties of pristimerin and celastrol isolated from Celastrus hypoleucus[J]. Pest Manag Sci, 2005,61(1):85-90
doi: 10.1002/ps.953 pmid: 15593077
[41] Tiedemann RE, Schmidt J, Keats JJ, Shi CX, Zhu YX, Palmer SE, Mao X, Schimmer AD, Stewart AK . Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-κB with antimyeloma activity in vitro and in vivo[J]. Blood, 2009,113(17):4027-4037
doi: 10.1182/blood-2008-09-179796
[42] Zhong L, Li B, Mah CS, Govindasamy L , Agbandje-McKenna M, Cooper M, Herzog RW, Zolotukhin I, Warrington KH Jr, Weigel-Van Aken KA, Hobbs JA, Zolotukhin S, Muzyczka N, Srivastava A.Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses[J]. Proc Natl Acad Sci U S A, 2008,105(22):7827-7832
doi: 10.1073/pnas.0802866105
[43] Aslanidi GV, Rivers AE, Ortiz L, Govindasamy L, Ling C, Jayandharan GR, Zolotukhin S , Agbandje-McKenna M, Srivastava A.High-efficiency transduction of human monocyte-derived dendritic cells by capsid-modified recombinant AAV2 vectors[J]. Vaccine, 2012,30(26):3908-3917
doi: 10.1016/j.vaccine.2012.03.079
[44] Dong B, Nakai H, Xiao W . Characterization of genome integrity for oversized recombinant AAV vector[J]. Mol Ther, 2010,18(1):87-92
doi: 10.1038/mt.2009.258 pmid: 2803017
[45] Lai Y, Yue Y, Duan D . Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome > or = 8.2 kb[J]. Mol Ther, 2010,18(1):75-79
doi: 10.1038/mt.2009.256
[46] Wu Z, Yang H, Colosi P . Effect of genome size on AAV vector packaging[J]. Mol Ther, 2010,18(1):80-86
doi: 10.1038/mt.2009.255
[47] Zhang T, Hu J, Ding W, Wang X . Doxorubicin augments rAAV-2 transduction in rat neuronal cells[J]. Neurochem Int, 2009,55(7):521-528
doi: 10.1016/j.neuint.2009.05.005 pmid: 19450628
[48] Denby L, Nicklin SA, Baker AH . Adeno-associated virus(AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation[J]. Gene Ther, 2005,12(20):1534-1538
doi: 10.1038/
[49] Besse B, Planchard D, Veillard AS, Taillade L, Khayat D, Ducourtieux M, Pignon JP, Lumbroso J, Lafontaine C, Mathiot C, Soria JC . Phase 2 study of frontline bortezomib in patients with advanced non-small cell lung cancer[J]. Lung Cancer, 2012,76(1):78-83
doi: 10.1016/j.lungcan.2011.09.006 pmid: 22186627
[50] Kim GP, Mahoney MR, Szydlo D, Mok TS, Marshke R, Holen K, Picus J, Boyer M, Pitot HC, Rubin J, Philip PA, Nowak A, Wright JJ, Erlichman C . An international, multicenter phase II trial of bortezomib in patients with hepatocellular carcinoma[J]. Invest New Drugs, 2012,30(1):387-394
doi: 10.1007/s10637-010-9532-1
[51] Wu S, Chen JJ, Kudelka A, Lu J, Zhu X . Incidence and risk of hypertension with sorafenib in patients with cancer: a systematic review and meta-analysis[J]. Lancet Oncol, 2008,9(2):117-123
doi: 10.1016/S1470-2045(08)70003-2
[52] Wang S, Liu K, Wang X, He Q, Chen X . Toxic effects of celastrol on embryonic development of zebrafish(Danio rerio)[J]. Drug Chem Toxicol, 2011,34(1):61-65
doi: 10.3109/01480545.2010.494664 pmid: 20954803
[53] Kim HJ, Park GM, Kim JK . Anti-inflammatory effect of pristimerin on lipopolysaccharide-induced inflammatory responses in murine macrophages[J]. Arch Pharm Res, 2013,36(4):495-500
doi: 10.1007/s12272-013-0054-1
[54] Sassa H, Kogure K, Takaishi Y, Terada H . Structural basis of potent antiperoxidation activity of the triterpene celastrol in mitochondria: effect of negative membrane surface charge on lipid peroxidation[J]. Free Radic Biol Med, 1994,17(3):201-207
doi: 10.1016/0891-5849(94)90075-2
[55] Figueiredo JN, R®z B, Séquin U . Novel quinone methides from Salacia kraussii with in vitro antimalarial activity[J]. J Nat Prod, 1998,61(6):718-723
doi: 10.1021/np9704157
[56] Avilla J, Teixido A, Velazquez C, Alvarenga N, Ferro E, Canela R . Insecticidal activity of Maytenus species(Celastraceae) nortriterpene quinone methides against codling moth, Cydia pomonella(L.)(Lepidoptera: tortricidae)[J]. J Agric Food Chem, 2000,48(1):88-92
doi: 10.1021/jf990008w
[57] Lu Z, Jin Y, Chen C, Li J, Cao Q, Pan J . Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl[J]. Mol Cancer, 2010,9:112
doi: 10.1186/1476-4598-9-112
[58] Jayandharan GR, Aslanidi G, Martino AT, Jahn SC, Perrin GQ, Herzog RW, Srivastava A . Activation of the NF-κB pathway by adeno-associated virus(AAV) vectors and its implications in immune response and gene therapy[J]. Proc Natl Acad Sci U S A, 2011,108(9):3743-3748
doi: 10.1073/pnas.1012753108
[59] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D . Global cancer statistics[J]. CA Cancer J Clin, 2011,61(2):69-90
doi: 10.3322/caac.v61:2
[60] El-Serag HB . Hepatocellular carcinoma[J]. N Engl J Med, 2011,365(12):1118-1127
doi: 10.1056/NEJMra1001683
[61] Chen WQ, Zheng RS, Zhang SW . Liver cancer incidence and mortality in China, 2009[J]. Chin J Cancer, 2013,32(4):162-169
doi: 10.1111/1759-7714.12025 pmid: 26767022
[62] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E , Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, H®ussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J; SHARP Investigators Study Group.Sorafenib in advanced hepatocellular carcinoma[J]. N Engl J Med, 2008,359(4):378-390
doi: 10.1056/NEJMoa0708857
[63] Cheng B, Ling C, Dai Y, Lu Y, Glushakova LG, Gee SW , McGoogan KE, Aslanidi GV, Park M, Stacpoole PW, Siemann D, Liu C, Srivastava A, Ling C.Development of optimized AAV3 serotype vectors: mechanism of high-efficiency transduction of human liver cancer cells[J]. Gene Ther, 2012,19(4):375-384
doi: 10.1038/gt.2011.105
[64] Kota J, Chivukula RR , O’Donnell KA, Wentzel EA, Montgomery CL, Hwang HW, Chang TC, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, Mendell JT.Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model[J]. Cell, 2009,137(6):1005-1017
doi: 10.1016/j.cell.2009.04.021 pmid: 19524505
[65] Wu MC . Traditional Chinese medicine in prevention and treatment of liver cancer: function, status and existed problems[J]. J Chin Integr Med, 2003,1(3):163-164
doi: 10.3736/jcim
[66] Li M, Qiao C, Qin L, Zhang J, Ling C . Application of traditional Chinese medicine injection in treatment of primary liver cancer: a review[J]. J Tradit Chin Med, 2012,32(3):299-307
doi: 10.1016/S0254-6272(13)60029-1
[67] Zhai XF, Chen Z, Li B, Shen F, Fan J, Zhou WP, Yang YK, Xu J, Qin X, Li LQ, Ling CQ . Traditional herbal medicine in preventing recurrence after resection of small hepatocellular carcinoma: a multicenter randomized controlled trial[J]. J Integr Med, 2013,11(2):90-100
doi: 10.3736/jintegrmed2013021
[68] Yu H, Zhu GY, Xu RZ, Niu HZ, Lu Q, Li GZ, Wang ZY, Zhang DS, Gu N, Teng GJ . Arterial embolization hyperthermia using As2O3 nanoparticles in VX2 carcinoma-induced liver tumors[J]. PLoS One, 2011,6(3):e17926
doi: 10.1371/journal.pone.0017926
[69] Qu FL, Hao XZ, Qin SK, Liu JW, Sui GJ, Chen Q, Qu T, Zhang HP, Sun Y . Multicenter phase II clinical trial of arsenic trioxide injection in the treatment of primary hepatocarcinoma[J]. Zhonghua Zhong Liu Za Zhi, 2011,33(9):697-701
[70] Yi H, Du BY, Tan YH, Liu AJ, Luo H, Luo XX, Su JF, Wang HF . Joint therapeutic effect of hepatocarcinoma suicide gene therapy combined with Liuwei Dihuangwan on expression of connexin in vitro[J]. Zhongguo Shi Yan Fang Ji Xue Za Zhi, 2011,17(12):114-118
[71] Wang CJ, Li JJ, Chen ZB, Liu YZ, Xu XM, Deng L, Pan JQ . Chinese herbs of invigorating the spleen and removing blood stasis(JPHY) improve killing effects of CD/TK double suicide genes on human hepatocellular carcinoma[J]. Xian Dai Sheng Wu Yi Xue Jin Zhan, 2008,8(9):1605-1607
[1] Yu-fan Shen, Xue Zhang, Ying Wang, Fan-fan Cao, Georges Uzan, Bin Peng, Deng-hai Zhang. Celastrol targets IRAKs to block Toll-like receptor 4-mediated nuclear factor-κB activation. Journal of Integrative Medicine, 2016, 14(3): 203-208.
[2] Yuan-hui Zhang, Yuan Wang, Yusufali Ali Hussein, Frederick Ashby, Daniel Zhang, Zi-fei Yin, George V. Aslanidi, Arun Srivastava, Chang-quan Ling, Chen Ling. Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo. Journal of Integrative Medicine, 2014, 12(6): 483-494.
[3] Chang-quan Ling​, Li-na Wang, Yuan Wang​, Yuan-hui Zhang​, Zi-fei Yin, Meng Wang​, Chen Ling​. The roles of traditional Chinese medicine in gene therapy. Journal of Integrative Medicine, 2014, 12(2): 67-75.
[4] Chang-jun Wang, Jian-jun Li, Xiao-min Wen, Li Deng. Effect of recombinant adenoviruses with CD/TK fusion suicide gene on human hepatocellular carcinoma cells. Journal of Chinese Integrative Medicine, 2004, 2(1): 42-45.
Full text



[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Hao Li, Ming-jiang Yao, Wen-ming Zhao, Jie Guan, Lin-lin Cai, Ling Cui. A randomized, controlled, double-blind trial of Huannao Yicong capsule in senile patients with mild cognitive impairment. Journal of Chinese Integrative Medicine, 2008, 6(1): 25-31
[3] Jun Cai, Hua Wang, Sheng Zhou, Bin Wu, Hua-rong Song, Zheng-rong Xuan. Effect of Sijunzi Decoction and enteral nutrition on T-cell subsets and nutritional status in patients with gastric cancer after operation: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2008, 6(1): 37-40
[4] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[5] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[6] SUN Ming-yu, ZUO Jian, DUAN Ji-feng, HAN Jun, FAN Shi-ming, ZHANG Wei, ZHU Li-fang, YAO Ming-hui. Antitumor activities of kushen flavonoids in vivo and in vitro. Journal of Chinese Integrative Medicine, 2008, 6(1): 51-59
[7] Wei Zhang, Xiang-feng Lu, Xiao-mei Zhang, Jian-jun Wu, Liang-duo Jiang. A rat model of pulmonary fibrosis induced by infusing bleomycin quickly through tracheal intubation. Journal of Chinese Integrative Medicine, 2008, 6(1): 60-67
[8] Hai-feng Wei, Bai-liu Ya, Ling Zhao, Cui-fei Ye, Li Zhang, Lin Li. Evaluation of tongue manifestation of blood stasis syndrome and its relationship with blood rheological disorder in a rat model of transient brain ischemia. Journal of Chinese Integrative Medicine, 2008, 6(1): 73-76
[9] A-gao Zhou, Yong Zhang, Gang Kui, De-Yun Kong, Hai-liang Ge, Qiu-hua Ren, Jia-rong Dong, Sheng Hong, Xu-ming Mao, Yin Wang, Hui-zheng Zhang, Shu-jun Wang. Influence of traditional Chinese compound recipes with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 77-82
[10] Guo-hong Yuan, Xiao-jing Pang, He-chao Ma. Synergic effects of Danggui Buxue Decoction in reducing toxicity of cytoxan in tumor-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 83-88