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Pathways of cell death in response to HIV protease cocktail Ritonavir/Lopinavir in primary hepatocytes

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Pathways of cell death in response to HIV protease cocktail Ritonavir/Lopinavir in primary hepatocytes

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Content Pathways of Cell Death in Response to HIV
Protease Cocktail Ritonavir/Lopinavir in
Primary Hepatocytes
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2015
Jay Hu
Acknowledgements
I would like to thank my mentors, Drs. Cheng Ji and Vijay Kalra for their support
and guidance during my time at USC. To my former labmates Rhema and Hui: thank
you for all the help, the long nights, and the time spent bouncing ideas off each other.
To my current labmates Caryn and Chen, thank you for giving me a new home in the
same building. Lastly, thank you Dr. Danenberg for agreeing to serve on my committee
on such short notice.
A special piece of gratitude goes to Dr. Zoltan Tokes for giving me the opportunity to be
a part of the Master's program.
1
Table of Contents
List of Figures....................................................................................................... 3
Abstract................................................................................................................. 4
Background........................................................................................................... 5
Materials and Methods......................................................................................... 8
Drugs........................................................................................................... 8
Cells and Culture......................................................................................... 8
Protein Analysis........................................................................................... 9
Immunocytochemistry.................................................................................. 10
Results................................................................................................................... 12
Non Parenchymal Cells are not affected by HIV PI...................................... 12
HIV PI and EFV cause cyototoxicity in MPH................................................. 14
Antioxidant Response is Imparied by HIV PI......................................…....... 17
Discussion.............................................................................................................. 22
References..............................................................................................................27
2
List of Figures
Figure 1: Cell death staining of HSC
Figure 2: ER stress related protein markers on hepatic stellate cell.
Figure 3: Does dependent cell death in MPH
Figure 4: Protein expression of primary hepatocytes with 22.5 ug/mL HIV protease
treatment.
Figure 5: Dose dependent protein loss after HIV PI treatment
Figure 6: Western blots on nuclear proteins after nuclear/ cytoplasmic separation.
Figure 7: Downstream effectors of Nrf2 signaling
Figure 8: Immunocytochemistry comparison of Nrf2 and actin expression in mouse
primary hepatocytes treated with HIV PI
Figure 9: Quantification of positive Nrf2 expression in nuclei of mouse primary
hepatocytes
3
Abstract
Highly Active Anti Retroviral Therapy (HAART) has become an integral part of modern
Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS)
treatment. Liver damage is a common side effect of HAART, the mechanisms of which are
largely unknown. In this study, Ritonavir/Lopinavir cocktail was used to simulate HAART on
primary mouse cells to explore three possible hypotheses of HAART induced liver damage.
Activation of hepatic stellate cells (HSC) is responsible for liver fibrosis. Mouse primary
HSCs were treated with 22 ug/mL of Ritoanvir/Lopinavir, no cell death or activation was
present. Primary mouse hepatocytes displayed widespread cell deatlh upon treatment with
22ug/mL of Ritonavir/Lopinavir. Western blotting revealed marked decrease in common
housekeeping proteins GAPDH and B-actin. Further experiments revealed a reduction of
Nrf2, a primary antioxidant activator, in the nuclei of treated cells. Nrf2 is transported to the
nucleus through the actin cytoskeleton where it binds upstream to the promoters of
Antioxidant Response Elements and activates them, initiating downstream antioxidant
activity. With the B-actin structure disrupted, Nrf2 cannot activate the downstream
Glutathione-S-Transferase pathway, and cell death occurs through unmitigated oxidative
stress. This was confirmed with immunoflouresence, where untreated cells remained Nrf2
positive in their nuclei, while Ritonavir/Lopinavir treated cells were Nrf2 negative. GAPDH is
an inducer of apoptosis when translocated to the nucleus by SIAH1; the possibilty of cell
death due to nuclear GAPDH presence was also investigated. No GAPDH was found in the
nuclear fragments of any cells. We conclude that HIV protease treatment impairs anti-
oxidant activity in mouse primary hepatocytes, leading to widespread cell death via
unchecked oxidative stress.
4
Background
Human Immunodeficiency Virus (HIV) is a constantly adaptive retrovirus that
causes Acquired Immunodeficiency Syndrome (AIDS) responsible for millions of deaths
since its discovery in 1981. In response to its rapidly mutating nature, therapy for HIV
has evolved from singular drugs to a cocktail of drugs focusing on multiple targets
simultaneously. This method is described as Highly Active Anti-Retroviral Therapy, or
HAART (Flexner 1998, Tejerina et al 2011).
Human Immunodeficiency Viral Protease Inhibitor (HIV PI) drugs are widely
prescribed as part of HAART in developed countries(Flexner 1998, Tejerina et al 2011).
This therapy dramatically increases the life expectancy of the HIV infected patient,
however hepatotoxic side effects are known to occur. The mechanisms behind these
side effects remain unclear(Senise et al 2011, Reust 2011). Previous studies in
myofibroblasts and adipocytes have shown that HIV PI treatment increases oxidative
stress due to overproduction of reactive oxygen species (ROS) (Touzet and Phillips
2010, Lagathu et al 2007, Auclair et al 2014). Nuclear factor (erythroid-derived 2)-Like
2 (Nrf2) is a transcription factor that regulates multiple Antioxidant Response Elements
(AREs), which serve as the primary defense pathways against ROS in eukaryotic cells.
Nrf2 is normally sequestered in the cytoplasm by Kelch like ECH associated protein 1
(Keap1), and ubiquitinated by Cullin 3, resulting in its subsequent transport to a
proteosome for degradation(Itoh et al 199, Kobayashi et al 2004). Under oxidative
stress conditions, the central cysteine residues in Keap1 are disrupted, resulting in a
conformational change that releases Nrf2(Yamamoto et al 2004, Sekhar et al 2010).
5
Nrf2 is then translocated through the actin cytoskeleton to the nucleus, where it forms a
heterodimer with a small Maf protein and binds to the upstream promoter of ARE genes
to activate them(Yamamoto et al 2004, Sekhar et al 2010). AREs activated by Nrf2
include Superoxide Dismutase, Glutathione S Transferse (GST), NADH quinone
oxidoreductase 1 (NQO1), UGT1A, and heme oxygenase, among others (Hayes et al
2000, Venugopal and Jaiswal 1996, Yueh and Tukey 2007).
In this study we explored the toxic effects of two HIV PI drugs commonly
perscribed as a cocktail: Ritonavir and Lopinavir in primary mouse cell culture. Studies
were done in both parenchymal mouse primary hepatocytes, as well as non-
parenchymal mouse hepatic stellate cells (HSC). HSC, also called perisinusoidal cells,
are myofibroblasts located in the space of Disse between the sinusoids and the
hepatocytes. HSC account for 5-8% of all liver cells, and under normal conditions are in
a quiescent state, characterized by a large star-shaped cytoplasm containing retinol
filled lipid droplets(Greets 2001). The function of quiescent HSC is not well known.
HSC become activated when the liver is damaged: an activated HSC loses its
namesake shape, and secretes the vitamin A and collagen from its lipid vesicles.
Activated HSC are responsible for scar tissue formation in cirrhosis (Winoau et al 2007,
Krizhanovsky et al 2008). In this study, the effect of HIV PIs to hepatic stellate cells was
minimal. Primary hepatocytes treated with clinical doses of HIV PI displayed
widespread cell death accompanied by marked depletion of Glucose Response Protein
78 (GRP78), GAPDH, and B actin. GAPDH is normally associated with the later steps
of glycolysis, however it can also serve as a cell-death signal when translocated into the
6
nucleus by Seven-in-abstentia 1 (SIAH1) (Hara et al 2005, Kang et al 2004). Actin is
involved in cytoplasmic structure, and plays a role in the transport of Nrf2 into the
nucleus for anti-oxidant protection (Kang et al 2004). We test two possible mechanisms
of HIV PI toxicity in hepatocytes: death by apoptosis via GAPDH nuclear translocation
or by oxidative stress from impaired ARE signaling. The results of testing illustrate
impaired antioxidant response as a possible mechanism for HIV PI induced
hepatotoxicity.
7
Materials and Methods
Drugs
Ritonavir/Lopinavir cocktail was used as the HIV Protease Inhibitor drug.
Efavirenz, an HIV nuclease inhibitor, was used as another drug in the HAART cocktail,
to verify the effects seen in the study are exclusive to HIV Protease drugs.
Tunicamycin was used as endplasmic reticulum stress control, as previous studies have
suggested that HIV PI causes cell death through ER stress (Kao et al 2012).
Cells and Culture
Primary hepatocellular stellate cells (HSC) were isolated from four month old
male C57BL/6 mice (Jackson Laboratory, Bar Harbor ME) by the Non-Parenchymal
Liver Cell Core (Southern California Research Center for Alcoholic Liver and Pancreatic
Disease), as previously described (Machida et al 2009). Cells were seeded onto
uncoated 10 cm petri dishes at roughly 1.5 million cells per dish. Accounting for the
30% attachment rate, the final cell count is estimated to around 0.5 million cells per
dish. The cells were allowed to attach and grow in glucose Dulbecco’s modified Eagle
Medium (DMEM, from Gibco/Life Technologies, Grand Island NY) with 10% Fetal
Bovine Serum for three days before treatment. Treatments were administered directly
to the medium on the third day at the following concentrations: 10 uL/mL DMSO as a
vehicle control, 7.5ug/mL of tunicamycin, 7.5uL/mL of 95% ethanol, 22.5ug /mL of
Ritonavir and Lopinavir, 22.5 ug/mL of Ritonavir, Lopinavir with 7.5 uL of 95% ethanol,
and 15 ug /mLof Efavirenz. Treatments lasted for twelve or twenty-four hours, at which
point the cells were stained for viability with Sytox green and Hoechst Blue and
8
visualized on a fluorescence microscope. Cells were then detached using 0.05%
Trypsin EDTA (Gibco/ Life Technologies, Grand Island NY), centrifuged at 3000 g and
washed with PBS.
Primary mouse hepatocytes were provided by the Cell Culture Core (USC
Research Center for Liver Disease) from 6 month old mice (Machida et al 2009, Ji et al
2011). Accounting for a 45% attachment rate, it is estimated that there will be 0.5
million cells per dish, the same amount as the HSC. Primary hepatocytes do not need
as much time to attach and activate, so the drug treatments were given on the second
day. The concentrations of the drugs used and time of treatment are identical to the
HSC, with the exception of the dose-response experiments which used drug
concentrations of 7.5ug, 11 ug, 22.5 ug, and 45 ug/mL.
Protein analysis
Nuclear and cytoplasmic separation was done through differential centrifugation.
After detaching the cells and washing three times with PBS, primary hepatocytes were
incubated in hypotonic lysis buffer (10 mM HEPES, 10mM KCL, 1.5mM MgCl2, 0.5 mM
DTT pH 7.9) for 5 minutes. This was followed by homogenization with a pestle to break
the cytoplasm of the swollen cells. The suspension was then transferred to a 0.25 M
sucrose solution and centrifuged at 1430rcf for 5 minutes to separate the nuclear
fragments from the cytoplasmic. The supernatant was kept as the cytoplasmic
fragment, while the pellet was re-suspended in a 0.88 M sucrose solution and
centrifuged at 3000rcf to purify the nuclear fragment.
9
The pellet obtained after centrifugation was then immersed in 150uL RIPA buffer
(Santa Cruz Biotechnology, Santa Cruz CA) and centrifuged at 20,000 rcf at 4 C for one
hour. The supernatant was removed post centrifugation and further concentrated using
Pierce PES concentrators with a 3K molecular weight cutoff (Thermo Scientific,
Rockford IL). Bradford protein assay (Bio-Rad Laboratories, Hercules CA) was
conducted to measure the total protein levels. 30ug of protein was mixed with Laemlli
sample buffer containing 2-Mercaptoethanol at a 1:1 volume ratio and loaded per well
on a mini-Protean TGX gel (Bio-Rad Laboratories, Hercules CA) and gel
electrophoresis was run at 38 constant amperes. The proteins were then transferred
onto a nitrocellulose membrane and probed with antibodies for GRP78, and
SarcoEndoplasmic Reticulum Calcuim 2+ ATPase (SERCA). Membranes were
visualized with Immunocruz Enhanced Chemiluminescence (Santa Cruz Biotechnology,
Santa Cruz CA) and exposed onto x-ray film.
Immunocytochemistry
Mouse primary hepatocytes were seeded onto microscope cover slips and
treated with DMSO, Tunicamycin, and HIV PI as previously mentioned. After 12 hours
of treatment, the cells were fixed in 5% buffered neutral formalin for 20 minutes. The
coverslips were blocked with 5% normal goat serum in PBS + 0.1% Triton X-100 for 1hr.
Coverslips were incubated in rabbit anti-Nrf2 antibody (Abcam, Cambridge MA) for one
hour, then probed with a rhodamine TRITC fluorescent antibody (Jackson
Immunoresearch, West Grove PA) for another hour. Filamentous actin double-staining
was done using Alexa Fluor 488 conjugated phalloidin (Life Technologies, Grand Island
10
NY). Nuclear counterstaining was done using Hoescht blue, and the coverslip was
mounted onto a glass slide and visualized on a Nikon Eclipse TE300 inverted
fluorescence microscope. A negative control with only rhodamine TRITC antibody
added without a primary was done to control for auto-fluorescence. Cells with co-
localized nuclear positive staining were counted across 3 slides at 20x magnification
and expressed as a percentage. Mean percentages were compared across treatments
and significance was calculated using the paired T test.
11
Results
Non-Parenchymal cells are not affected by HIV PI
Staining with Sytox Green/Hoescht blue reavealed that HSC treated with HIV PI
did not exhibit more cell death than control cells. Very little cell death was seen, and
morphology between control and experimental groups was identical (Figure 1).
12
Figure 1 Cell death staining of HSC: Compiled image showing Sytox Green and
Hoescht Blue staining of Hepatocellular stellate cells with various treatments. Top row,
from left to right: 45ug RitLop, 45 ug RitLop+ 15 uL 95% EtOH, 45 ug EFV. Bottom
row, from left to right: DMSO control, 5ug Tunicamycin, 12 ug 95% EtOH. No
difference in morphology or toxicity was observable between the treatments.
Western blot results from HSC treated with HIV PI suggest that there are no
significant changes in levels of ER stress markers as a result of treatment. GRP78
produced a strong band at 12 hours in all 3 HIV protease treated groups. CHOP was
only found in the Tunicamycin group. SERCA bands were even throughout all
treatments at 12 hours. B-actin was used as a loading control; bands even throughout
all groups (Figure 2).
13
Figure 2: ER stress
related protein markers
on hepatic stellate cell.
D: DMSO Vehicle
TM: Tunicamycin
A: Alcohol
RL: Ritonavir/Lopinavir
22.5 ug/mL
RLA: Ritonavir/Lopinavir
22.5 ug/mL + Alcohol
EFV: Efavirenz 15 ug/mL
HIV PI and EFV cause cytotoxicity in primary mouse hepatocytes
Microscopy results in primary hepatocytes reveal widespread cell death in MPH
cells when treated with HIV protease and nuclease inhibitors. Little to no Sytox green
staining was seen at any timepoint in the DMSO vehicle control, Tunicamycin, or
Alcohol treated groups, even when the concentrations were doubled, indicating low cell
death rates. All three HIV protease treatments at both the original and doubled drug
concentrations showed prolific Sytox green staining at 12 hours, these results were
mirrored in the 24 hour timepoint. Overlay images with DAPI indicate an 85-90% death
rate, along with cell clumping. In the HIV PI and HIV PI + Alcohol groups, the cytoplasm
and nuclei of the hepatocytes were distorted and irregularly shaped. In the EFV group,
the cells looked healthy and regularly shaped at a glance, but Sytox green positive
staining indicated that they were mostly dead. Experiments done at 1/3 of the standard
HIV protease dosage (15ug Ritoavir, 15ug Lopinavir, 15 ug EFV) did not display
extensive positive Sytox green staining. In these treated groups, the amount of cell
death was only slightly higher than the DMSO control (Figure 3).
14
15
Figure 3: Dose dependent cell death in MPH. Compiled image showing Sytox Green and
Hoescht Blue staining of various HIV Protease dosages on mouse primary hepatocytesat
12 hours. Labels are from left to right.
Top row: 45ug/mL RitLop, 45 ug/mL RitLop + 24uL 95% EtOH, 45 ug/mL EFV.
Second row: 22.5 ug/mL RitLop, 22.5ug/mL RitLop + 12 uL 95% EtOH, 22.5 ug/mL EFV.
Third row: 7.5 ug/mL RitLop, 7.5 ug/mL RitLop + Alcohol + 4uL 95% EtOH, 7.5 ug/mL EFV.
Fourth row: DMSO control , 5 ug Tunicamycin, 12 uL 95% EtOH
Western blots revealed widespread loss of multiple proteins in HIV PI treated
groups. In 6 month old male mice, GRP78 was depleted in the HIV PI (RitLop, RitLopA)
groups. SERCA faintly expressed in all treatments, but especially depleted in the HIV
PI treated groups. Caspase 3 was not expressed, indicating non-apoptotic death
pathway. Curiously, bands for the two standard housekeeping genes, B actin, and
GAPDH, completely disappeared in the RitLop and RitLopA groups, although they
appeared normally in the EFV treated group. PDI, a protein folding chaperone, was
used as a housekeeping protein instead to check for protein loading error. PDI was
evenly expressed across all lanes and treatments, indicating that the reduction of
GAPDH and B-actin expression was not a loading error (Figure 4).
16
Figure 4: Western blot showing protein expression of primary hepatocytes with 22.5
ug/mL HIV protease treatment.
A: PDI B: GRP78 C: GAPDH D: SERCA, E: Caspase 3
PDI expression is constant throughout, GAPDH and GRP 78 are selectively depleted.
In experiments where a lower 7.5 ug/mL dose of HIV protease was used,
GAPDH was faintly expressed at both 12 and 24 hour timepoints, as opposed to
completely undetectable in the 22.5 ug/mL and 45 ug /mL dose groups. PDI remained
consistently expressed across all treatment groups, dosages, and timepoints (Figure 5).
Antioxidant Response is Impaired by HIV PI
Western blots were performed on separated nuclear and cytosolic fractions of
MPH to detect the presence of Nrf2, AREs, and the presence of nuclear GAPDH.
Lamin H1, a nuclear envelope protein, was used as a nuclear control to further confirm
nuclear/cytoplasmic separation. Lamin B1 bands appeared in the nuclear fragment of
17
Figure 5: Dose dependent protein loss after HIV PI treatment. Western blot showing
PDI and GAPDH expression in primary hepatocytes based on HIV Protease dosage.
Left: GAPDH expression at increasing doses, compared to EFV and Tunicamycin
treatments.
Right: RitLop, RitLop + Alcohol vs EFV treatments.
A: PDI 45 ug/mL dose , B: GAPDH 45 ug/ mL dose, C: PDI 22.5 ug/mL dose
D: GAPDH 22.5 ug /mL dose E: PDI 7.5 ug/mL dose, F: GAPDH 7.5 ug/mL dose
all groups except HIV PI, and was absent from all cytoplasmic fractions. The band for
Nrf2 protein was also much less intense in the nuclear fraction of the HIVPI treated
groups (Figure 6). Two proteins induced downstream by Nrf2, Uridine Glycotransferase
1-A (UGT1A) and Glutathione-S-Transferase, were also tested to determine which
pathway, if any, was affected by the reduced Nrf2. UGT1A had even bands across all
treatments in the nuclear fraction and no bands in the cytoplasmic. GST bands had
reduced intensity in the PI treated groups in the cytoplasmic fragment, and no bands in
the nuclear fragment. Blots done after the separation of nuclei and cytoplasm revealed
no GAPDH to be present in the nuclei of any cells. Predictably, GAPDH bands were
observed in the cytoplasmic fraction of DMSO, Tunicamycin, and EFV treated groups,
but did not appear in the RitLop treated groups. Actin was tested not as a loading
control, but because it is involved in the translocation of Nrf2 to the nucleus, where Nrf2
begins its anti-oxidant signaling. Actin was found in both nuclear and cytoplasmic
fractions, but the bands were reduced or disappeared in the PI treated groups for both
fractions (Figure 7).
18
Figure 6: Western blots on nuclear proteins after nuclear/ cytoplasmic
separation. Nrf2 shows reduced expression in RitLop treated groups, but does
not affect UGT1A downstream expression.
Immunocytochemistry was done on primary mouse hepatocytes in order to
confirm the impairment of Nrf2 nuclear transport, Hepatocytes have inherently high
auto-fluorescence, as evidenced by the negative control having a co-localized red and
green color when stimulated with rhodamine (TRITC) and fluorescin (FITC)
wavelengths. Nuclei were free of auto-fluorescence, and without DAPI counter staining
showed as empty areas. Red Nrf2 rhodamine positive staining appeared in the nuclei
of DMSO control, Tunicamycin, and alcohol treated groups, co-localized to a purple
when counterstained with DAPI. In the HIV PI treated groups, the red nuclear Nrf2
staining was significantly reduced, and completely dissapeared in most areas. Green
staining actin filaments were generally long and fibrous in DMSO, Tunicamycin, and
Alcohol groups, but shorter and more disjointed in HIV PI treated groups (Figure 8).
19
Figure 7: Downstream effectors of Nrf2 signaling. UGT1A is undisturbed,
however GST expression is affected. GAPDH is not localized in the nucleus, but
cytoplasmic expression is still reduced in HIV PI treated groups.
Quantification of the nuclear co-localization of Nrf2 reveals over 90% of cells in DMSO
and Tm treated groups express Nrf2 positive staining, while less than 10% of HIV PI
treated cells show Nrf2 nuclear expression (Figure 9).
20
DMSO Tm HIV PI HIV PI + EtOH
0
20
40
60
80
100
120
Nuclear Nrf2 Expression

Treatment
% Cells w/Positive Nrf2
*
*
* P < 0.05
vs DMSO
Figure 9: Quantification of positive Nrf2 expression in nuclei of mouse
primary hepatocytes. Cell count was obtained by counting over 3 slides and
comparing means. * = P < 0.05 when compared to DMSO
Figure 8 (on next page): Immunocytochemistry comparison of Nrf2 and actin
expression in mouse primary hepatocytes treated with HIV PI and DMSO
vehicle control. Top Row: Nrf 2 immunofluoresence w/ rhodamine TRITC
secondary antibody. Second row: Actin staining using Alexa Fluor 488
conjugated phalloidin. Third row: Nuclear staining with DAPI. Fourth row:
Merged image showing co-localization of TRITC Nrf2 and DAPI in nuclei as
purple.
21
Discussion
HIV Protease inhibitors and nuclease inhibitors are frequently prescribed to AIDS
patients as part of HAART. As Ritonavir is often clinically prescribed to enhance the
effects of HIV PI, it was used in conjunction with Lopinavir in this study to represent
treatment with HIV PI (Flexner 1998, Tan and Walmsley 2007). Dosage was selected
based on previous studies; while it is shown that doses of 2.1ug/mL or
Ritonavir/Lopinavir was sufficient to inhibit protease activity in HIV-1 strains in peripheral
cells (Flexner 1998, Danner et al 1995, Justesen 2008), a higher dosage should be
used to properly simulate drug concentrations in the liver, being the primary site for drug
detoxification. In other studies, doses as high as 100uM have been used, as such the
range of 7.5 -45ug/mL is justified (Parker et al 2005, Zhou et al 2006).
Non-parenchymal HSC cells do not seem involved in HIV PI mediated
cytotoxicity. No cell death was seen in the Sytox green staining, and western blot
results of ER stress markers did not show significant differences from the controls. The
morphology between HIV PI, EFV, and control treated HSC was indistinguishable, with
no indication of activation. In all treatments, HSC retained their quiescent stellate
shaped cytoplasm, with numerous visible lipid vesicles. Given the lack of morphological
or cytotoxic effects, oxidative stress markers were not examined in HSC.
In mouse primary hepatocytes, 22.5ug/mL doses of both Ritonavir/Lopinavir
(HIV PI)and Efavirenz (HIV NI) caused widespread cytotoxicity, however the
morphology of the dead cells as well as downstream western blot analysis suggest that
these drugs induce cell death through different pathways. Previously published
22
literature has shown that cells without a Rho mitochondrial receptor show significantly
less cytotoxic reactivity to EFV, suggesting an method of action possibly through cellular
respiration pathways (Apostolova et al 2013, Apostolova et al 2010). The GAPDH, B-
actin depletion, alongside deformed cytoplasms of HIV PI induced cell death suggest
that the death pathway is a disruption of a cytoplasmic process. GAPDH expression
being lowered at the 7.5ug/mL dosage without cell death occurring suggests that the
loss of GAPDH is not a result of cell death, and possibly a cause or precursor to it.
Previous work by Hara et al indicates that GAPDH induces cell death and apoptosis
when translocated into the nucleus of the cell by the E3 ubiquitin ligase, Seven in
Absentia 1 (SIAH1) transporter. SIAH1 is normally a very transient protein, with an
intracellular half life of 5 minutes(Hara et al 2005, Hara et al 2006). GAPDH stabilizes
this protein when bound to it, and is trans-located through the nuclear membrane as a
complex; GAPDH cannot innately cross through the nuclear membrane otherwise.
When SIAH1 is in the nucleus, it begins to degrade nuclear proteins, causing cell death.
The nuclear/cytoplasmic separation experiments in the current study suggest that this
pathway is not the mechanism of cell death in MPH treated with HIV PI. No GAPDH
was detected in the nuclear fragments of any treatment group, therefore the loss of
cytoplasmic GAPDH is not due to being transported to the nucleus. It is reasonable to
conclude from these results that the hepatocellular cytotoxicity of Ritonavir/Lopinavir
HIV cocktail is not mediated by GAPDH-induced apoptosis.
Ruling out the possibility of GAPDH inducing apoptosis, the disappearance of
normally stable cytoplasmic proteins such as GAPDH, B-actin, GRP78, and SERCA
23
suggest a disruption of metabolism in HIV PI treated groups, possibly through
heightened oxidative stress. Supporting this hypothesis is the lack of an Nrf2 band in
the HIV PI treated groups. Nrf2 is a transcription factor that promotes transcription of
many genes required for defense against cellular antioxidants, and is considered the
primary defense against cytotoxic effects of oxidative stress. Nrf2 remains present in
control DMSO treated, Tunicamycin, Alcohol, and EFV treated groups. The Nrf2 band
in EFV was also present, continuing to suggest a different mechanism of cytotoxicity
from EFV. Given the results of the downstream effectors from Nrf2, it seems likely that
the cell death is caused by a loss of activity in the GST pathway, as there was no
difference in band intensity in the UGT1A immunoblots between drug treated and
control groups. The question remains: does HIV PI treatment cause increased
oxidative stress that overwhelms the anti-oxidant defenses resulting in cell death, or
does it disrupt antioxidant pathways and thus allowing normal levels of oxidative stress
result in cytotoxicity? Actin binds to Keap1, causing it to release Nrf2 translocate it to
the nucleus where it is activates transcription of numerous AREs. The loss of actin in
both nuclear and cytoplasmic fractions may suggest that actin structure is disrupted in
the cytoplasm, resulting in an inability to transport NRF2. This may further result in
lowered antioxidant activity, causing cell death through unchecked oxidative stress.
Cellular staining with immunocytochemistry supports this hypothesis. Nrf2 is widely
expresed in over 90% of all nuclei except when treated with HIV PI, where the
expression falls under 10%. Phalloidin based actin staining is not visibly reduced in HIV
PI groups , however the long, filamentous characteristics are more disjointed and not as
24
pronounced in groups treated with HIV PI. These results lend visual confirmation to the
loss of Nrf2 in the nuclei of HIV PI affected cells.
This is the first study investigating the role of HSC in liver damage caused by HIV
PI drugs. The exact role of HSC is unclear, but they are known to play a role in the
formation of liver fibrosis. In healthy livers HSC are generally in the quiescent state, but
become activated during liver injury. Activation can be recognized by a morphological
change (loss of the trademark “star shaped” cytoplasm) and results in chemotaxis, and
upregulation of wound healing pathways. Results involving HSC are negative. HIV PI
did not induce cell death, nor did it accelerate activation of HSC.
A previous study in this lab established the role of alcohol exacerbating ER
stress in mouse primary hepatocytes (Kao et al 2012), through regulation of calcium
iron homeostasis via SERCA inhibition, there was no indication of a widespread protein
depletion, with full B-actin bands. This previous study utilized 6 week old mice, which
are still in adolescence. The current study utilizes 6 month old mice to more accurately
represent a young adult. Studies on alcohol and aging have described that while
adolescents are more sensitive to the neurosedative effects of alcohol than adults, that
there is no major difference in liver adaptation between the two groups (Wood et al
1986, Quoilin et al 2013). It should be noted that in these studies, 6 month old mice
were considered “young”, with 28 month old mice in the “aged” group. Curiously, there
is a lack of ER stress or alcohol enhanced toxicity in contrast to the previous study,
however there is a noticeable shift towards oxidative stress. Studies have shown that
aging affects responses to oxidative stress, specifically in reduced GST levels (Vogt and
25
Richie 2007). This study proposes a possible mechanism of hepatic cytotoxicity caused
by HIV PI, and shows a marked reduction in Nrf2 and GST expression in cells treated
with HIV PI. Given the number of patients that are prescribed HAART, these results
may be useful in reducing the occurrence of HIV PI related liver complications. Further
studies may be needed to show if simultaneous treatment with antioxidants can
attenuate HIV PI induced cell-death and restore Nrf2 nuclear expression. The pathways
for UGT1A and GST may also be more clearly illustrated in further study, as the loss of
Nrf2 affects only GST expression. In conclusion, the results of this study suggest an
oxidative stress mediated mechanism for HIV protease inhibitor induced cytotoxicity of
mouse primary hepatocytes.
26
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29

Abstract (if available)

Abstract Highly Active Anti Retroviral Therapy (HAART) has become an integral part of modern Human Immunodeficiency Virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS) treatment. Liver damage is a common side effect of HAART, the mechanisms of which are largely unknown. In this study, Ritonavir/Lopinavir cocktail was used to simulate HAART on primary mouse cells to explore three possible hypotheses of HAART induced liver damage. Activation of hepatic stellate cells (HSC) is responsible for liver fibrosis. Mouse primary HSCs were treated with 22 ug/mL of Ritonavir/Lopinavir, no cell death or activation was present. Primary mouse hepatocytes displayed widespread cell death upon treatment with 22ug/mL of Ritonavir/Lopinavir. Western blotting revealed marked decrease in common housekeeping proteins GAPDH and B-actin. Further experiments revealed a reduction of Nrf2, a primary antioxidant activator, in the nuclei of treated cells. Nrf2 is transported to the nucleus through the actin cytoskeleton where it binds upstream to the promoters of Antioxidant Response Elements and activates them, initiating downstream antioxidant activity. With the B-actin structure disrupted, Nrf2 cannot activate the downstream Glutathione-S-Transferase pathway, and cell death occurs through unmitigated oxidative stress. This was confirmed with immunofluorescence, where untreated cells remained Nrf2 positive in their nuclei, while Ritonavir/Lopinavir treated cells were Nrf2 negative. GAPDH is an inducer of apoptosis when translocated to the nucleus by SIAH1

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Creator Hu, Jay (author)

Core Title Pathways of cell death in response to HIV protease cocktail Ritonavir/Lopinavir in primary hepatocytes

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 06/22/2015

Defense Date 06/22/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ER stress,hepatocytes,HIV protease inhibitor,Nrf2,OAI-PMH Harvest,oxidative stress

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kalra, Vijay K. (committee chair), Danenberg, Peter (committee member), Ji, Chen (committee member)

Creator Email hujay@usc.edu,jayswho@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-574755

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