Fasudil is a potent inhibitor for various protein kinases such as myosin light chain kinase and protein kinase C. It has been used as a drug for improvement of intracranial vasospasm and following ischaemic diseases. In this report, we demonstrate that fasudil suppressed the replication of human immunodeficiency virus type 1 (HIV-1) in mitogen-activated peripheral blood mononuclear cells. Our finding shows that fasudil may be useful as a new and distinct chemotherapeutic agent against HIV-1 infection

Adachi, Akio

Akari, Hirofumi

Hiwada, Hiroko

Koyama, A. Hajime

Saito, Yasuyoshi

Takayanagi, Noriyasu

Yamamoto, Yoshihiko

Tokushima University Institutional Repository

INTRODUCTIONFasudil (1-(5-isoquinolinesulphonyl)-homopiperazine) isa newly developed drug which improves intracranialischemia as a result of the dilating effect of vasospasm (1,2). The neuroprotective property of fasudil is due to itseffect that inhibits the activities of various protein kinasesincluding myosin light chain kinase, cyclic AMP-dependentprotein kinase, cyclic GMP-dependent protein kinase andprotein kinase C (3, 4).Previous studies have shown that H-7, a protein kinaseinhibitor, inhibits the activation of transcription of humanimmunodeficiency virus type1 (HIV-1) long terminal repeat(5) and HIV-1 replication in chronically infected cell lines(6). These effects of H-7 are caused by blockage of theprotein kinase C-dependent cascade (5, 6). The similarefficacy of H-7 and fasudil as kinase inhibitors led to anidea that fasudil inhibits HIV-1 replication. If so, fasudilwould be a candidate as a new anti-HIV-1 agent whosemechanism is different from that of currently availableanti-HIV-1 drugs targeted for viral reverse transcriptase(RT) and protease. To address this issue, we examined thepotential suppressive effect of fasudil on the replication ofHIV-1 in vitro .MATERIALS AND METHODSCellsA human cervical cell line, HeLa, was maintained inDulbecco's modified Eagle's medium supplemented with10% heat-inactivated fetal calf serum. CD4-positive humanlymphocytic cell lines, CEMx174, Jurkat, M8166, andMT-4,were maintained in RPMI 1640 medium supplementedwith 10% heat-inactivated fetal calf serum. Human peripheralblood mononuclear cells (PBMCs) were prepared andused as previously described (7).Cell proliferation assayCell proliferation was evaluated by the modified MTTmethod designated WST-1 as indicated in the manufacture'sinstruction (Dojindo Laboratories, Kumamoto, Japan).Transfection and infectionFor transfection, uncleaved plasmid DNA designatedpNL432 (an infectious molecular clone of HIV-1) (8) wasintroduced into HeLa cells by the calcium-phosphatecoprecipitation method (8). CD4-positive cell lines andPBMCs were infected with cell-free virus samples preparedfrom transfected HeLa cells as previously described (9).RT assayVirion-associated RT activity was measured as describedpreviously (10).RESULTSCytotoxicity of fasudil for T cell lines and PBMCsAs a first step to monitor the effectiveness of fasudil(Asahi Chemical Industry Co., Ltd., Shizuoka, Japan)against HIV-1, its cytotoxicity for various cell types wasevaluated (Fig.1). In a human CD4+ leukemia cell line,Jurkat, obvious cytotoxic effects were observed whenfasudil was added to the culture at concentrations higherthan 100µM, and no cytotoxicity was detected at ̃37.5 µMof fasudil (Fig.1 A). Similar results were obtained in theother human T cell lines, CEMx174, M8166, and MT-4(data not shown). In phytohemagglutinin P (PHA-P) (DifcoLaboratories, Detroit, USA.)-stimulated PBMCs, the cytotoxicSuppression of HIV-1 replication in peripheral blood mononuclearcells by fasudilHirofumi Akari＊, Yoshihiko Yamamoto＊, Hiroko Hiwada＊, Yasuyoshi Saito†,Noriyasu Takayanagi†, A. Hajime Koyama＊, and Akio Adachi＊＊Department of Virology, The University of Tokushima School of Medicine, Tokushima, Japan ; and †Institute forLife Science Research, Asahi Chemical Industry Co. Ltd., Shizuoka, JapanAbstract : Fasudil is a potent inhibitor for various protein kinases such as myosin light chain kinaseand protein kinase C. It has been used as a drug for improvement of intracranial vasospasm andfollowing ischaemic diseases. In this report, we demonstrate that fasudil suppressed the replicationof human immunodeficiency virus type 1 (HIV-1) in mitogen-activated peripheral blood mononuclearcells. Our finding shows that fasudil may be useful as a new and distinct chemotherapeutic agentagainst HIV-1 infection. J. Med. Invest. 44 : 211-214, 1998Key Words : fasudil, protein kinase inhibitor, HIV-1Received for publication December 24, 1997 ; accepted January 12, 1998.１ Address correspondence and reprint requests to Akio Adachi, Ph.D.,Department of Virology, The University of Tokushima School of Medicine,Kuramoto-cho, Tokushima 770-8503, Japan and Fax : +81-886-33-7080.The Journal of Medical Investigation Vol.44 1998２１１dose of fasudil was basically similar to that inJurkat cells. In activated PBMCs cultivatedfor 24 hours in complete medium (RPMI-1640medium with 10% fetal calf serum, L-glutamine,2-mercaptoethanol and antibiotics) (CM) (Fig.1B) and for 15 days in CM supplemented with50U/ml human recombinant interleukin-2 (rIL-2)(Serotec Ltd., Oxford, England) (Fig.1 C), thecytotoxic effect was seen at concentrationshigher than 100 µM of fasudil.Effect of fasudil on HIV-1 replication in T celllines and PBMCsOn the basis of the cytotoxic data of fasudildescribed above, concentrations below 50 µMwere used for the inhibition assay of virusreplication.First, the effect of fasudil on HIV-1 replicationin T cell lines was monitored. As shown inFig.2, in the two cell lines (CEMx174 andM8166), the virus growth kinetics in thepresence (5 µM) or absence of fasudil were surprisinglysimilar. In a repeated experiment, essentially similarresults were obtained (data not shown). Furthermore,cultivation of the two cell lines infected with various dosesof virus in the presence of higher concentration of fasudil(50 µM) gave no significant effects with respect to virusproduction at 14 days post-infection (data not shown).We then asked whether fasudil is effective against HIV-1growth in PBMCs, the natural target cells. Fig.3 showsthe replication kinetics of HIV-1 in PHA-P-stimulatedPBMCs cultivated in rIL-2-supplemented CM in thepresence of various concentrations of fasudil. On the peakday of RT production, the activity was reduced by 75%,50%, 34%, and 33% of that of a control (no fasudil) atconcentrations of 40, 20, 10, and 5 µM of fasudil, respec-tively. In a repeated experiment, essentially similar resultswere obtained (data not shown).DISCUSSIONOur results here showed that fasudil has a suppressiveeffect on HIV-1 growth in activated PBMCs but not inestablished T cell lines. The basis for this observation isunclear. It has been shown that cell activation is requiredfor HIV-1 replication in primary T lymphocytes (11, 12).Fasudil could inhibit the activity of protein kinase(s)required for HIV-1 replication in PBMCs but not in T celllines. It is possible that fasudil affects the function of thenef gene which is known to be important for the viralreplication in PBMCs (13, 14) and disease progression(15-17). A recent finding that fasudil inhibits Nef-dependentcytotoxicity (18) may be related to its suppressive effect ofHIV-1 replication reported here.Apart from the mechanism for the inhibitory effect, ourresults show that fasudil has a potential as a candidatefor an anti-HIV-1 agent which can be administrated inFig.1. Cytotoxicity of fasudil for human lymphocytic cells. Jurkat cells (A) and unstimulated PBMCs (B) were cultured in CM in the presenceof fasudil for 48 and 24 hours, respectively, and cell proliferation was evaluated by the WST-1 method. (C) PBMCs activated with PHA-P (1 µg/ml) were cultured in CM with 50 U/ml of rIL-2 and fasudil for 15 days (half volume of the medium was changed every 3 days), and the cellgrowth was measured as above. Triplicate samples were analyzed (absorbance at 450 nm), and the averages with error bars are shown.Fig.2. Effect of fasudil on HIV-1 replication in established cell lines. Cell-freeviruses (3 x 10５ RT units) obtained from HeLa cells transfected with pNL432 wereinoculated into 2 x106 cells of CEMx174 (A) and M8166 (B), and RT productionin the culture supernatants was monitored at indicated intervals. Symbols : ○,mock-infection ; ●, virus growth in the absence of fasudil ; △, virus growth in thepresence of 5 µM of fasudil.H. Akari et al. Effect of fasudil on HIV-1 replication２１２combination with inhibitors of the viral RT or protease. Tofurther explore the possibility of fasudil as an anti-HIV-1drug, animal experiments using simian immunodeficiencyvirus/macaque model system are to be done.ACKNOWLEDGEMENTSWe thank Kazuko Yoshida for editorial assistance. Thiswork was supported by grants-in-aid for AIDS researchfrom the Ministry of Education, Science and Culture ofJapan, and the Ministry of Health and welfare of Japan.REFERENCES1. Shibuya M, Suzuki Y, Sugita K, Saito I, Sasaki T,Takakura K, Nagata I, Kikuchi H, Takamae T, HidakaH, NakashimaM : Effect of AT877 on cerebral vasospasmafter aneurysmal subarachnoid hemorrhage. J Neurosurg76 : 571-577, 19922. Satoh S, Suzuki Y, Harada T, Ikegaki I, Asano T,Shibuya M, Sugita K, Hidaka H : Possible prophy-lactic potential of HA1077, a Ca2+ antagonist andvasodilator, on chronic cerebral vasospasm. Eur JPharmacol 220 : 243-248, 19923. 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JVirol 65 : 3514-3520, 19918. Adachi A, Gendelman HE, Koenig S, Folks T, WilleyR, Rabson A, Martin MA : Production of acquiredimmunodeficiency syndrome-associated retrovirus inhuman and non-human cells transfected with aninfectious molecular clone. J Virol 59 : 284-291, 19869. Folks T, Benn S, Rabson A, Theodore T, HogganMD,Martin M, Lightfoote M, Sell K : Characterization of acontinuous T-cell line susceptible to the cytopathiceffects of the acquired immune deficiency syndrome(AIDS)-associated retrovirus. Proc Natl Acad Sci USA82 : 4539-4543, 198510. Willey RL, Smith DH, Laskey LA, Theodore TS,Earl PL, Moss B, Capon DJ, Martin MA : In vitromutagenesis identifies a region within the envelopegene of the human immunodeficiency virus that iscritical for infectivity. J Virol 62 : 139-147, 198811. Stevenson M, Stanwick TL, Dempsey MP, LamonicaCA : HIV-1 replication is controlled at the level of Tcell activation and proviral integration. EMBO J 9 :1551-1560, 199012. Bukrinsky MI, Stanwick TL, Dempsey MP, StevensonM : Quiescent T lymphocytes as an inducible virusreservoir in HIV-1 infection. Science 254 : 423-427,199113. Miller MD, Warmerdam MT, Gaston I, Greene WC,Feinberg MB : The human immunodeficiency virus-1nef gene product : a positive factor for viral infectionand replication in primary lymphocytes and macrophages.J Exp Med 179 : 101-113, 199414. Spina CA, Kwoh TJ, Chowers MY, Guatelli JC,Richman DD : The importance of nef in the inductionof human immunodeficiency virus type 1 replicationfrom primary quiescent CD4 lymphocytes. J ExpMed 179 : 115-123, 199415. Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL,Desrosiers RC : Absence of intact nef sequences in along-terminal survivor with nonprogressive HIV-1infection. N Engl J Med 332 : 228-232, 199516. Mariani R, Kirchhoff F, Greenough TC, Sullivan JL,Desrosiers RC, Skowronski J : High frequency ofdefective nef alleles in a long-term survivor withnonprogressive human immunodeficiency virus type 1infection. J Virol 70 : 7752-7764, 199617. Premkumar DRD, Xue-Zhong MA, Maitra RK,Fig.3. Effect of fasudil on HIV-1 replication in PBMCs. The activatedPBMCs were infected with cell-free NL 432 prepared from transfectedHeLa cells at a multiplicity of infection of 0.01. Virus titer wasdetermined in MT-4 cells. The cultures were maintained in CM with50 U/ml of rIL-2 and 40 µM(□), 20 µM(◇), 10 µM(△), 5 µM(▽),or absence (●) of fasudil. RT production in the culture supernatantswas monitored at indicated intervals. +, mock-infection.２１３The Journal of Medical Investigation Vol.44 1998Chakrabarti BK, Salkowitz J, Yen-Lieberman B, HirschMS, Kestler HW : The nef gene from a long-term HIVtype 1 nonprogressor. AIDS Res Hum Retroviruses12 : 337-345, 199618. Okada H, Takei R, Tashiro M : Nef protein of HIV-1induces apoptotic cytolysis of murine lymphoid cellsindependently of CD95 (Fas) and its suppression byserine/threonine protein kinase inhibitors. FEBS Lett417 : 61-64, 1997H. Akari et al. Effect of fasudil on HIV-1 replication２１４

Suppression of HIV-1 replication in peripheral blood mononuclear cells by fasudil

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Suppression of HIV-1 replication in peripheral blood mononuclear cells by fasudil

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