AIDS RESEARCH AND HUMAN RETROVIRUSES

Volume 13, Number 4, 1997

Mary Ann Liebert, Inc.

Lecithinized Superoxide Dismutase: An Inhibitor of Human

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Immunodeficiency Virus Replication

MARIAPPAN PREMANATHAN,1 HIDEKI NAKASHIMA,1 RIE IGARASHI,2 YUTAKA MIZUSHIMA,2
and KANEO YAMADA2

ABSTRACT

Superoxide dismutase (SOD) is an enzyme used in the treatment of oxygen radical-related diseases.
AIDS Research and Human Retroviruses 1997.13:283-290.

Lecithinization of SOD enhances its pharmacological activity. Lecithinized SOD (PC-SOD) inhibits human
immunodeficiency virus (HIV) types 1 and 2 in MT-4 cells. HIV-1-infected MT-4 cells were cultured for 5
days in the presence of PC-SOD, at various concentrations. In an MTT assay, reverse transcriptase (RT) ac-
tivity of the cell extract and p24 antigen production were measured. Untreated, HIV-1-infected MT-4 cells
served as control. PC-SOD inhibited viral replication most effectively at 2500 U/ml, a concentration that did
not affect cell viability, with an EC50 value of 718 U/ml. PC-SOD treatment inhibited RT activity and p24
production in a dose-dependent manner. Western blot analysis of the HIV-1-infected MT-4 cells treated with
PC-SOD at 2500 U/ml did not detect any expression of viral proteins. Failure to inhibit virus adsorption,
proviral DNA and mRNA synthesis, and RT and proteinase enzyme activity suggests that the mechanism of
action of PC-SOD is entirely different from those of the currently available anti-HIV drugs. PC-SOD shows
synergistic interaction with AZT, ddl, ddC, KNI-272, and dextran sulfate. PC-SOD also inhibited the oxida-
tive stress-induced depletion of sulfhydryls, which are the cause of diminished antioxidant defenses in III V
infected patients.
-

INTRODUCTION Lecithinization of SOD potentiates its cell membrane affinity,

cellular permeability, and pharmacological activity.4'5

Acquired Immunodeficiency Syndrome (AIDS) caused by

the human immunodeficiency virus (HIV) has remained a
health threat of global significance. Because of the limitation
of currently available drugs, an extensive search for new anti-
HIV agents is ongoing. Most of the currently available antivi-
ral drugs show mitochondrial toxicity.1 The mitochondrial tox-
icity of AZT (zidovudine) is due to mutant mitochondrial DNA
caused by oxygen radicals.2 Free radicals can increase the repli-
cation of HIV and destroy immunocompetent cells such as T
cells.
Superoxide dismutase (SOD) is an enzyme that catalyzes the
dismutation of the reactive and potentially harmful free radi-
cals to less toxic hydrogen peroxide and molecular oxygen.
Various attempts have been made to use it in the treatment of
oxygen radical-related diseases. It has been shown that HeLa
cells transfected with HIV tat gene markedly suppress the ex-
pression of Superoxide dismutase. Diminished antioxidase de-
fenses cause the rapid depletion of plasma sulfhydryls.3
Currently, lecithinized SOD (PC-SOD) is undergoing clinical
evaluation for oxidative diseases. To provide a rationale for
combination therapy with PC-SOD, we have investigated the
effect of PC-SOD alone and in several combination ratios with
the currently available anti-HIV drugs and its inhibitory reac-
tivity on the radical-induced depletion of plasma sulfhydryls.
MATERIALS AND METHODS
Reagents and chemicals
The following reagents were obtained from the indicated
companies: dextran sulfate (8 kDa) (Kowa, Tokyo); AZT,
dideoxyinosine (ddl), and dideoxycytidine (ddC) (Yamasa
Shoyu Co., Chiba, Japan); KNI-272 (Japan Energy, Tokyo);
RPMI 1640 medium (GIBCO, Grand Island, NY); fetal calf
serum (FCS) (Whittaker Bioproducts, Walkersville, MD); 3-

1
Department of Microbiology and Immunology, Kagoshima University School of Dentistry, Kagoshima-Shi 890, Japan,
institute of Medical Science, St. Marianna University, Kawasaki, Kanagawa 216, Japan.

283
 284
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Wako
Pure Chemicals, Osaka, Japan); recombinant human CuZn-
SOD (rhCuZn-SOD) (Ube Kosan Co., Ltd., Yamaguchi, Japan).
PC-SOD, in which four molecules of a phosphatidylcholine
(PC) derivative were covalently bound to each dimer of
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rhCuZn-SOD,45 and potassium peroxochromate (^CrOs),6
were synthesized as described earlier.
Cells and viruses
A human T lymphotropic virus type I (HTLV-I) positive T
cell line, MT-4,7 and lymphoblastoid T cell line, MOLT-4
(clone No. 8),8 were subcultured twice a week at a concentra-
tion of 3 X 105 cells/ml in RPMI 1640 medium supplemented
with 10% (v/v) heat-inactivated FCS. A strain of HIV-1 tub was
prepared from the culture supernatant of MOLT-4/HIV-lnm
cells that were persistently infected with HIV-lnm. A strain of
HIV-2rod was prepared from the culture supernatant of MT-4
cells infected with HIV-2ROd- Clinical isolates of HIV-lAoi2B
AIDS Research and Human Retroviruses 1997.13:283-290.
and HIV-lAoi2D (AZT resistant) were also maintained in MT-
4 cell cultures.
MTT assay
The inhibitory effect of the rhCuZn-SOD and PC-SOD on
HIV-1 replication was monitored by the inhibition of virus-in-
duced cytopathogenicity in MT-4 cells. Briefly, MT-4 cells
were suspended at 3 X 105 cells/ml and infected with HIV-1 at
a multiplicity of infection (MOI) of 0.01. The HIV-infected or
mock-infected MT-4 cells were placed in 96-well microtiter
plates (200 /xl/well) and incubated at 37°C in a C02 incubator
in the presence of the compound. After 5 days, cell viability
was quantified by the MTT assay, as described previously,9,10
from which the 50% cytotoxic concentration (CC50), 50% ef-
fective concentration (EC50), and selectivity indices (SI =
CC50/EC50) were calculated.
Assay for viral expression
MT-4 cells were treated with virus for 90 min for the ad-
sorption of virus. After adsorption, the unbound virus was re-
moved by repeated washing with the medium and then resus-
pended in medium containing various concentrations of
PC-SOD and incubated at 37°C. After 5 days of incubation, the
number of viable cells was monitored by the trypan blue dye
exclusion method and HIV-1 antigen-positive cells were mon-
itored by indirect immunofluorescence using serum from an
AIDS patient and fluorescein isothiocyanate (FITC)-labeled
anti-human IgG antibody.
Detection of HIV-1 p24gag antigen
Cell-free culture supernatant of MT-4 cells with or without
PC-SOD treatment was collected and the presence of p24 anti-
gen was detected and quantified by the HIV-1 p24 core profile
enzyme-linked immunosorbent assay (ELISA), using the method
described by the manufacturer (Abbott GmbH Diagnostika,
Wiesbaden-Delkenheim, Germany). Briefly, the standards were
run in the range of 12.5 to 100 pg/ml and the antigen-antibody
complex was probed with a streptavidin-horseradish peroxide
(HRP) conjugate. The end product was quantified by the inten-
PREMANATHAN ET AL.
sity of the color, which is directly proportional to the amount of
HIV-1 p24 core antigen captured. Color development was read
at 492 nm, using a colorimeter.
Syncytium formation assay
MOLT-4 cells (5 X 105) were cultured with an equal num-
ber of the MOLT-4/HIV-1 urn cells in microtiter plate wells con-
taining various concentrations of PC-SOD. After 24 hr of cocul-
tivation, the number of giant cells (syncytium) was recorded by
microscope examination and the fusion index was calculated as
described earlier.11
Virus adsorption assay
The inhibitory effect of PC-SOD on virus adsorption was
measured by an indirect immunofluorescence-laser flow cyto-
fluorographic method.12 MT-4 cells were exposed to a high con-
centration of HIV-1 virions in the presence or absence of PC-
SOD. The PC-SOD was added 1 min before the virus was
added. The cells were incubated for 1 hr at 37°C and washed
twice in phosphate-buffered saline (PBS) to remove the unab-
sorbed virus. A high-titer polyclonal antibody derived from a
patient with AIDS-related complex (diluted 1:500 in PBS) was
then added. After 1 hr of incubation at 37°C, the cells were
washed twice with PBS. The cells were then incubated with
FITC-conjugated F(ab')2 fragments of rabbit anti-human im-
munoglobulin antibody (diluted 1:30 in PBS) for 1 hr at 37°C,
washed twice in PBS, resuspended in 1 ml of 0.5%
paraformaldehyde in PBS, and analyzed by laser flow cytoflu-
orography.
Reverse transcriptase assay
A reverse transcriptase (RT) assay was done by 3H-based
RT scintillation proximity, using the Quan-T-RT assay system
(Amersham International pic, Buckinghamshire, England)13
with recombinant HIV-1 RT enzyme (Seikagaku Co., Tokyo).
Briefly, primer-template/beads, thymidine 5'-triphosphate
(TTP)/[3H]TTP, recombinant RT enzyme, and PC-SOD were
mixed in an appropriate concentration and incubated at 37°C.
After 1 hr, reaction was terminated by stop reagent and diluted
with Tris-buffered saline (10 mM Tris-HCl [pH 7.4] and 0.15
M NaCl) and counted by a scintillation counter.
Reverse transcriptase enzyme activity of the supernatant and
cell extract of the HIV-infected MT-4 cells was assayed as de-
scribed earlier.14,15 The cell cultures were harvested after 5 days
and separated from their supernatants by centrifugation. The
cell pellets were solubilized by vigorous vortexing in a lysis
buffer containing 0.2% Triton X-100 in 25 mM Tris-HCl (pH
7.4). The supernatants and the solubilized samples were ana-
lyzed for RT enzyme activity as described above.
Proteinase enzyme assay
The HIV-1 proteinase enzyme assay was done by means of
a 125I-based scintillation proximity assay (SPA), using an HIV
proteinase (125I)-SPA enzyme assay kit that is under develop-
ment at Amersham International, with recombinant HIV-1 pro-
teinase enzyme (AGMED, Inc., Bedford, MA). The 125I-labeled
SPA beads, recombinant HIV proteinse enzyme, and PC-SOD
were mixed in an appropriate concentration and incubated for
 ANTI-HIV ACTIVITY OF LECITHINIZED SOD
2 hr at room temperature. The reaction was terminated
reagent and counted by a scintillation counter.
Western blot analysis
The presence of HIV-1 antigens in the culture supernatant
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of MT-4 cells with or without PC-SOD treatment was deter-
mined by Western blot analysis. MT-4 cells infected with HIV-
Ihib and uninfected control cells were cultured for 6 days in
the presence of various concentrations of PC-SOD. After 6
days, cell-free supernatant was centrifuged at 36,000 rpm at 4°C
for 1 hr in an ultracentrifuge and the pellet was used for Western
blot assay. Viral proteins were denatured and separated by 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred onto a Hybond-ECL (nitrocellu-
lose) membrane filter.16 Western blot analysis was performed
by the standard protocol, using an ECL Western blot kit
(Amersham International) with HIV-positive serum as primary
antibody and biotinylated antibody as second antibody.17
AIDS Research and Human Retroviruses 1997.13:283-290.
Polymerase chain reaction amplification
MT-4 cells were treated with virus and incubated for 90 min
at 37°C for virus adsorption. The cells were then washed three
times with medium and further incubated with media contain-
ing different concentrations of PC-SOD. After 12 hr, total
mRNA and DNA were isolated from 107 cells. mRNA and DNA
were extracted by standard protocol, using a DYNAL
Dynabeads mRNA direct kit (Dynal AS, Oslo, Norway) and an
IsoQuick (ORCA Research, Inc., Bothell, WA) nucleic acid ex-
traction kit, respectively. Reverse transcriptase-based poly-
merase chain reaction (RT-PCR) was performed for mRNA am-
plification by use of a GeneAmp rTth reverse transcriptase RNA
PCR kit (Perkin-Elmer/Roche Molecular Systems, Inc.,
Branchburg, NJ) and primers SK38 and SK39 (upstream and
downstream primers of the gag gene sequence of HIV). For
DNA amplification, each reaction mixture contained 10 mM
Tris-HCl (pH 8.8), 50 mM KC1, 1.5 mM MgCl2, 0.1% Triton
X-100, a 0.25 mM concentration of each of the four dNTPs,
SK38 and SK39 primers (0.1 pM each), and 2.5 U of Taq DNA
polymerase enzyme (Wako Pure Chemicals), overlaid with min-
eral oil and amplified in a thermal cycler (Perkin-Elmer DNA
thermal cycler).10
Sulfhydryl determination
Both nonprotein and protein sulfhydryls were quantified by
disulfide exchange with DTNB at pH 8.18 Plasma (100 /¿l) was
added to 100 pi of sodium dodecyl sulfate (10%, w/v) and
mixed thoroughly. Eight hundred microliters of phosphate
buffer (5 mM, pH 8.0) was added and the background absorp-
tion read at 412 nm. The solution was then incubated for 1 hr
at 37°C in the presence of 100 pi of DTNB (0.4 mg/ml). The
resulting thioquinone was measured in a photometer at 412 nm.
Reduced glutathione was used for calibration.
Analysis of drug combination effect
The inhibition of HIV-1 replication by combination of PC-
SOD and AZT, ddl, ddC, KNI-272, or dextran sulfate was eval-
uated in experiments involving multiple concentration ratios of
the drugs. For an experiment, different concentrations of each
285
by stop drug, drug combinations, were assayed in a checkerboard
or
manner. Antiviral activity in combination drug-treated HIV-in-
fected MT-4 cells was determined by the protection against
HIV-induced cytopathic effect (CPE) assessed by the MTT
method as described above. The combination indices (CIs) were
evaluated by three-dimensional analysis19 using a Macintosh
computer (Apple Computer, Inc., Cupertino, CA) with a
Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA)
and a Deltasoft graphics program (Delta Point, Inc., Monterey,
CA).
RESULTS
Anti-HIV assay
When rhCuZn-SOD and PC-SOD were evaluated for their in-
hibitory effect on the cytopathogenicity of HIV-1 in MT-4 cells
by MTT assay, rhCuZn-SOD did not show any activity (Fig.
1 A). On the other hand, PC-SOD completely protected the cells
against virus-induced cell destruction at a concentration of 1250
U/ml. It showed a dose-dependent inhibition of HIV-1 with a
mean 50% effective concentration (EC50) and EC90 values of
718.1 and 1009.9 U/ml, respectively (Fig. IB). A 50% cytotox-
icity (CC50) was observed at the concentration of 5217.1 U/ml.
Anti-HIV activity of PC-SOD was observed with several strains
of both HIV-1 and HIV-2 in MT-4 cells. Irrespective of the cri-
teria used to assess anti-HIV activity, i.e., inhibition of viral cy-
topathogenicity, antigen expression, RT activity, and p24 pro-
duction, PC-SOD invariably inhibited HIV-1, including
AZT-resistant strain, and HIV-2 replication within the EC50 con-
centration range of 422 to 846 U/ml (Table 1 ). For comparison
we have also tested the anti-HIV activity of synthetic pseudo
PC-SOD, which contains inactive SOD instead of active SOD,
and did not observe any activity against HIV (data not shown).
PC-SOD inhibited viral expression in MT-4 cells in a dose-
dependent manner as measured by immunofluorescence (IF)
staining (Fig. IB) and reduced the amount of p24 in the cul-
ture supernatant of MT-4 cells infected with HIV-1 (Fig. 2).
The results were determined from the standard curve of known
amounts of p24 antigen and expressed in terms of nanograms
of p24 per milliliter of supernatant.
The effect of different concentrations of PC-SOD on HIV-1
replication was quantitated by RT assay. Figure 3 shows the
RT values from cell extracts of HIV-1-infected MT-4 cells with
various concentrations of PC-SOD. The RT value for each sam-
ple was calculated from triplicate cultures and compared with
that of the infected control culture (100%). The EC50 was cal-
culated from the graph. These results are in agreement with the
MTT assay.
Virus adsorption
Various experiments were undertaken to elucidate the mech-
anism of action of PC-SOD. First we investigated whether PC-
SOD inhibited the binding of HIV particles to MT-4 cells, as
assessed by laser flow cytometry. PC-SOD inhibited HIV ad-
sorption weakly, by only 35% at a concentration of 5000 U/ml.
However, when we investigated the inhibitory activity against
multinuclear giant cell (syncytium) formation in cocultures of
persistently HIV-1-infected MOLT-4 cells (MOLT-4/HIV-
 286 PREMANATHAN ET AL.

PC-SOD
(U/ml)
5000

2500
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1250

mock infected
cell control
O 78.13 156.25 312.5 625 1250 2500 5000
SOD ( U/ml ) 0 100 200 300

p24 in culture supernatant (ng/ml)

FIG. 2. Inhibition of HIV-1 p24 expression in infected MT-
4 cells by PC-SOD. Infected MT-4 cells were incubated in the
presence or absence of different concentrations of PC-SOD for
AIDS Research and Human Retroviruses 1997.13:283-290.

5 days. The expression of p24 antigen was measured by HIV-

1 p24 core profile enzyme-linked immunosorbent assay
(ELISA). All experiments were conducted in triplicate. The av-
erage concentration of p24 was calculated from the culture su-
pernatant, using the standard curve, and expressed in nanograms
per milliliter.

Iihb) and uninfected MOLT-4 cells, PC-SOD efficiently in-

625 1250
78.13 156.25 312.5
PC-SOD (U/ml)
FIG. 1. (A and B) Anti-HIV activity and viral antigen inhibi-
tion in MT-4 cells by unmodified rhCuZn-SOD and PC-SOD,
respectively. The viability of HIV-infected MT-4 cells (black
columns) and mock-infected MT-4 cells (white columns) was
measured by the MTT method 5 days after infection. The num-
ber of viable cells was expressed as the percentage of mock-in-
fected drug-free control cells. HIV-1 antigen-positive cells were
detected by indirect IF and laser flow cytometry, using a poly-
clonal antibody as a probe. The number of viral antigen-positive
cells was expressed as a percentage of the HIV-infected drug-
free control cells. Each experiment was performed at least three
rimes and the results are the means of the three experiments.
hibited syncytium formation with an EC50 value of 1884.36
U/ml.
Reverse transcriptase and proteinase enzyme assay
Reverse transcriptase and proteinase enzymes could be ex-
cluded as a target for PC-SOD, because it did not cause a
marked reduction in the activity of recombinant RT and re-
combinant proteinase enzymes.
Western blot
We examined HIV-1 protein synthesis in cells treated with
PC-SOD. Western blotting of a viral pellet from cell-free su-

Table 1. Wide-Spectrum Anti-HIV Activity of PC-SODa

Virus
and Day of EC50 CC50
strain Cells Assay analysis (U/ml) (U/ml) SI

HIV-1
IIIB MT-4 MTT 718.08 38.39 5217.06 ± 189.25 7.26
IIIB MT-4 p24 antigen 842.66 53.34
IIIB MT-4 RT 708.33 32.16
A012B MT-4 Ag expression 422.45 19.77
A012Db MT-4 Ag expression 845.38 38.64
HIV-2
ROD MT-4 MTT 801.10 ± 33.24 5308.67 ± 143.48 6.63

"The EC50 is calculated on the basis of the inhibition of HIV-induced cytopathogenicity, or the reduction of p24 antigen in the
culture supernatant, or the inhibition of RT activity in lysed cellular extract, or HIV antigen expression in MT-4 cells. The CC50
is calculated on the basis of the reduction of the viability of mock-infected cells. Data represent the mean values with standard
deviations for at least three separate experiments. SI, Selectivity index (CC5o/EC50); Ag, antigen.
bAZT-resistant HIV-1.
 ANTI-HIV ACTIVITY OF LECITHINIZED SOD
120
100
a were oxidized in a cell-free system in the presence of K3Q-O8.21
o
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à?
PC-SOD (U/ml)
FIG. 3. Inhibition of HIV-1 reverse transcriptase by PC-SOD
AIDS Research and Human Retroviruses 1997.13:283-290.
treatment. Lysed cellular extracts from the control and experi-
mental MT-4 cell cultures were collected after 5 days of incu-
bation and assayed for reverse transcriptase as described in text.
All samples were assayed in triplicate, and the values were ex-
pressed as a percentage of the control values.
pernatant of cells infected for 6 days exhibited a typical HIV-
1 protein pattern (Fig. 4). Almost all viral protein bands disap-
peared when the sample was treated with 2500 U of PC-SOD
per milliliter and reduction in HIV proteins was seen when the
sample was treated with lower concentrations.
Polymerase chain reaction
To determine whether PC-SOD inhibited the replication of
proviral DNA and mRNA of HIV-1, we analyzed proviral DNA
and mRNA of cell extracts by polymerase chain reaction (PCR)
using SK38 and SK39 primers in the gag gene sequence. There
is no inhibition in proviral DNA and mRNA production.
71K-
43K-
28K-
18K^
~. JÊL.
i 7
FIG. 4. Western blot. Identification of HIV-1 antigens in MT-
4 cells with or without PC-SOD treatment. Lane 1, control HIV
sample for Western blot; lane 3, experimental control HIV sam-
ple; lanes 4, 5, and 6, PC-SOD treatment (625, 1250, and 2500
U/ml, respectively); lane 7, cell control.
287
Oxidative stress-dependent depletion of
plasma sulfhydryls
The redox status of plasma sulfhydryls is a sensitive marker
of oxidative stress.20 Both nonprotein and protein sulfhydryls
The concentration of sulfhydryls in plasma of a healthy indi-
vidual averages around 600 pM, while that of AIDS patients
is dramatically depressed to 250 pM.22 Whether PC-SOD is
able to inhibit the oxidant-induced K3Cr08-dependent deple-
tion of thiols was tested ex vivo in the plasma of healthy vol-
unteers. A 1 mM concentration of K3Cr08 was required to ox-
idize plasma sulfhydryls to the level observed in HIV patients
and PC-SOD at 300 U/ml totally inhibited the oxygen radical-
dependent sulfhydryl depletion (Fig. 5).
Combination study
The effects of combinations of PC-SOD with different drugs
on HIV-1 replication in MT-4 cells were also studied. We first
investigated the antiviral effect of PC-SOD combined with
AZT. HIV-infected MT-4 cells were incubated with serially di-
luted AZT together with various concentrations of PC-SOD.
After 5 days, the cell viability was measured by MTT assay and
the viable cell number was plotted (experimental dose-response
curve in Fig. 6A). The theoretical additive effect (Fig. 6B) was
calculated directly from the individual dose-response curves
and synergy plot (Fig. 6C), yielded by subtracting the additive
curve from the experimental curve. The amount of synergy ob-
served with combinations of the two compounds is represented
by the height of the bars in the graph when the percentage of
interaction is plotted versus drug concentrations. As shown in
Fig. 6C, a combination of PC-SOD and AZT in the dose range
of 100 to 200 U/ml and 0.025 to 0.05 pM, respectively, was
consistently more effective than the use of either drug alone,
because the plots appear above the calculated additivity. A syn-
S
0 4-
75 150 300 Serum
control
PC-SOD ( U/ml )
FIG. 5. Inhibition of peroxochromate-induced depletion of
plasma sulfhydryls by PC-SOD. Human plasma sulfhydryls
were oxidized with 1 mM K3CrOg in the presence and absence
of PC-SOD (hatched columns). Sulfhydryl groups were deter-
mined by disulfide exchange with DTNB at pH 8. The data are
presented as means of triplicate experiments.
 288
1„
g=:
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> tivity, and the clear inhibition of viral protein as detected by
¡E
c
< of the currently available AIDS drugs. The comparable effi-
O-' J*"« "^
B cytoid cells suggests a role for PC-SOD as an antiviral agent.
AIDS Research and Human Retroviruses 1997.13:283-290.
5 by oxygen radicals.2 PC-SOD efficiently scavenged Superoxide
e anión, and increased the cell membrane affinity and pharma-
< cologie potency of SOD.5 It increases the cellular antioxidase
a
FIG. 6. Three-dimensional analysis of anti-HIV interactions
between PC-SOD and AZT. Viability of HIV-infected MT-4
cells is expressed as percentage of mock-infected and untreated
control cells. (A) Experimental dose-response curve. (B)
Additivity calculated from the dose-response curve of PC-SOD
and AZT alone. (C) Synergy plot, yielded by subtracting the
additive curve (B) from the experimental curve (A).
ergistic effect was observed with other drugs (ddl, ddC, KNI-
272, and dextran sulfate) (Fig. 7), particularly the combinations
of drugs at concentrations nearer to their EC50 values (e.g., AZT
at 0.025 to 0.05 pM, ddl at 6.25 to 25 pM, ddC at 0.625 to 2.5
/xM, KNI-272 at 1 to 2 pg/ml, and dextran sulfate at 0.25 to
0.5 /u,g/ml). PC-SOD showed a synergistic interaction with all
of the drugs at a concentration of 200 and 100 U/ml, which is
three to six times lower than the EC50 value.
DISCUSSION
The results presented in this article demonstrate that PC-SOD
interferes with HIV replication in MT-4 cells. PC-SOD com-
pletely inhibited HIV-1 infection and cytopathogenecity in MT-
PREMANATHAN ET AL.
4 cells at optimal concentration. This protection is directly re-
lated to viral inhibition, and is not due to cellular toxicity of
the PC-SOD. This is also confirmed by an [3H]thymidine up-
take experiment (data not shown) and by a tetrazolium-based
colorimetric assay for viable cells (Fig. IB) after 5 days of in-
cubation with PC-SOD. Detection of proviral DNA and mRNA
by PCR analysis, the failure of RT and proteinase enzyme ac-
Western blot (Fig. 4) suggest interference with later events in
the virus life cycle—an entirely different mechanism from those
ciency of PC-SOD in inhibiting syncytium formation in mono-
Specific in vitro activity of rhCuZn-SOD by the xan-
thine-xanthine oxidase method was 3467 U/mg whereas that of
PC-SOD was 2876 U/mg (equivalent to 83% of rhCuZn-SOD).4
In our study rhCuZn-SOD did not show any activity whereas
PC-SOD showed activity. Most of the currently available an-
tiviral drugs show mitochondrial toxicity.1 The mitochondrial
toxicity of AZT is due to mutant mitochondrial DNA caused
defense system and inhibits the oxidative stress-dependent de-
pletion of plasma sulfhydryls (Fig. 5). Both intra- and extra-
cellular sulfhydryls are critically lowered in HIV seroposi-
tives.23,24 The inorganic compound K3CrOs is stable at alkaline
pH, but decays readily to Superoxide, hydrogen peroxide, hy-
droxyl radicals, and singlet oxygen at physiological pH, the
same oxidants that are produced by activated phagocytes and
that are the cause of diminished antioxidant defenses in HIV-
infected patients suffering from frequent opportunistic infec-
tions.22 The use of this inorganic mimic in a cell-free system
allows the clear distinction of oxidant-induced sulfhydryl oxi-
dation from non-reactive oxygen species (ROS)-dependent
mechanisms.
The pandemic threat of HIV-1 infection has prompted an in-
tensive search for new antiviral agents. Considering the sever-
ity of AIDS and the current necessity for long-term chemother-
apy, more effective and less toxic drugs are needed. In
combination experiments PC-SOD showed good synergistic in-
teraction. A synergistic effect was observed with drugs AZT,
ddl, ddC, KNI-272, and dextran sulfate, particularly combina-
tions of these drugs at concentrations nearer to their EC50 val-
ues (Fig. 7). It is reasonable to see an enhancement in antivi-
ral activity in the combination study with the other available
anti-HIV drugs, as it acts on an entirely different target. Clinical
evaluation indicated that combination therapy with AZT and
ddC was more effective than therapy with single agents.25 For
this drug combination, clinical results appeared to correlate with
in vitro data obtained in cell culture experiments on synergy.26
The PC-SOD is likely to be an important supportive drug for
combination trials.
Anti-HIV activity of Cu2Zn2 SOD has been reported.18
Antiviral effects of SOD have also been demonstrated in vari-
ous murine and human cell lines.27 It was shown that HeLa
cells transfected with HIV tat gene markedly suppress the ex-
pression of Superoxide dismutase. Diminished antioxidase de-
fenses cause the rapid depletion of sulfhydryls.3 PC-SOD is ef-
fective in blocking HIV infection and its replication in T cells,
 ANTI-HIV ACTIVITY OF LECITHINIZED SOD
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C
O
03
-
0>
OJD
Q
AIDS Research and Human Retroviruses 1997.13:283-290.
FIG. 7. Synergy plot. Interactions between PC-SOD and (A) ddl, (B) ddC, (C) KNI-272, and
inhibits the oxidative stress-dependent depletion of plasma
sulfhydryls, synergistically interacts with other AIDS drugs,
and deserves further evaluation in terms of a potential combi-
nation drug treatment of AIDS as based on our earlier results.
Until the clinical results are known, caution should be used in
extrapolating in vitro findings to the in vivo situation because
of the involvement of complex host and viral factors, such as
viral burden and existence of viral reservoirs.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, and Culture
of Japan, and the Japan Health Sciences Foundation. One of the
authors (M.P.) is grateful to the Japanese Foundation for AIDS
Prevention, Japan for financial support.
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Address reprint requests to:
Hideki Nakashima
Department of Microbiology and Immunology
Kagoshima University School of Dentistry
8-35-1 Sakuragaoka
Kagoshima-Shi 890, Japan