|

Neurolipidomics.org

Apoptosis (2007) 12:969–977DOI 10.1007/s10495-007-0755-3 HIV protease inhibitors modulate apoptosis signaling in vitro and
in vivo
Stacey R. Vlahakis · Steffany A. L. Bennett ·
Shawn N. Whitehead · Andrew D. Badley
Published online: 9 February 2007
Springer Science + Business Media, LLC 2007 Abstract HIV protease inhibitors are an integral part of
Keywords Apoptosis . HIV protease inhibitors . Neurons .
effective anti-HIV therapy. The drugs block HIV protease, prevent proper packaging of HIV virions, and decrease theHIV viral burden in the peripheral blood of infected individ- HIV infection causes a progressive depletion of CD4+ T uals. In addition to direct anti-viral effects, the HIV protease cells. There are likely many reasons for the CD4+ T cell inhibitors also modulate apoptosis. A growing body of work loss, including apoptosis. Current therapy for HIV-infected demonstrates the anti-apoptotic effects of HIV protease in- patients often includes a combination of reverse transcriptase hibitors on CD4+ and CD8+ T cells during HIV infection.
inhibitors and HIV protease inhibitors. The first HIV protease The mechanism of this apoptosis inhibition is supported by inhibitor was FDA approved for anti-HIV therapy in humans several proposed hypotheses for how they alter the fate of in 1995. Currently, there are ten HIV protease inhibitors that the cell, including preventing adenine nucleotide translo- are approved for clinical use in humans (saquinavir, indi- cator pore function, which consequently prevents loss of navir, darunavir, nelfinavir, lopinavir, amprenavir, atazanavir, mitochondrial transmembrane potential. More recently, the tipranavir, ritonavir, and fosamprenavir). These drugs are in- anti-apoptotic effects of the HIV protease inhibitors have hibitors of the HIV viral aspartyl protease. The compounds been tested in non-HIV, non-immune cell, whereby protease have a strong affinity for the active site of the HIV pro- inhibitors prevent apoptosis, and disease in animal models of tease and irreversibly inhibit the catalytic activity of the sepsis, hepatitis, pancreatitis and stroke. Interestingly, when HIV protease inhibitors are used at supra-therapeutic con- When HIV protease is inhibited, viral particles are pro- centrations, they exert pro-apoptotic effects. This has been duced, but they are immature, and do not package prop- demonstrated in a number of tumor models. Although it is erly into infectious virions. Moreover, naturally occurring unclear how HIV protease inhibitors can induce apoptosis HIV protease mutations, which arise during suboptimal an- at increased concentrations, future research will define the tiretroviral therapy, result in impaired replication kinetics targets of the immunomodulation and reveal the full clinical of progeny virions. In addition to its role in viral replica- potential of this intriguing class of drugs.
tion, HIV protease may also contribute to HIV pathogenesis.
When transfected into a human or bacterial cell, HIV pro-tease is cytotoxic and causes cleavage of a variety of hostproteins including actin, Bcl2 and procaspase 8. It remainsunclear how HIV protease initiates cell death.
S. R. Vlahakis · A. D. Badley (
) Although the HIV protease inhibitors have limited Division Infectious Disease, Mayo Clinic College of Medicine,200 First Street SW, Rochester, MN 55905 bioavailability and stability, they are the basis of effec- tive anti-HIV therapy, and in combination with other an-tiretroviral agents, produce a sustained decrease in HIV viral S. A. L. Bennett · S. N. Whitehead load. Over the past ten years, evidence has accumulated that Neural Regeneration Laboratory, Department of Biochemistry,Microbiology and Immunology, University of Ottawa, HIV protease inhibitors have non-viral effects on the host 451 Smyth Road, Ottawa, ON, Canada, K1M 1E5 cells beyond the effect of blocking HIV protease enzymatic

Apoptosis (2007) 12:969–977 activity. For example, HIV protease inhibitors also directly significantly when a patient is treated with HIV PI containing affect cellular apoptosis signaling.
HAART, which correlates with a decrease in HIV peripheralblood viral load and an increase in peripheral blood CD4+T cells. In addition, CD4+ and CD8+ T cells isolated from The role of HIV protease inhibitors during treatment of
HIV-infected individuals before and on days 1, 4, and 8, after starting treatment with HIV PI containing HAART, haddecreased sensitivity to Fas-mediated apoptosis after being Optimal anti-HIV therapy is called highly active antiretrovi- on HAART for as little as one day ral therapy (HAART). This includes the combination of HIV A recent trial by Landay et al. focused on the mecha- reverse transcriptase inhibitors, often with an HIV protease nism behind HIV protease inhibitor immune effects. The inhibitor. Since the advent of HAART in late 1995, these reg- trial compared patients with suppressed HIV viral replica- imens have been shown to increase CD4+ T cell counts and tion on a PI-based anti-HIV regimen or non-PI based drug reduce the amount of HIV virus quantitated in the peripheral regimen. One week after enrollment in the study, subjects in blood, in infected individuals. However, in many cases, the the PI containing arm had less spontaneous T cell apoptosis increase in CD4+ T cells that occurs during HIV therapy than those in the non-PI containing arm. Although there are appeared to result from actions that were independent of the likely multiple reasons for CD4+ T cell decline during the effect of the drug on viral replication. In one such instance, course of HIV infection, these data suggest that patients on HIV-infected subjects were randomized in a clinical trial to HIV protease inhibitors have less spontaneous CD4+ T cell receive therapy with three drugs, including one protease in- apoptosis and have improved CD4+ T cell counts, further hibitor (saquinivir, zidovudine, and zalitabine) or two drugs.
suggesting that the HIV protease inhibitors block CD4+ T One of the two drug regimens included a protease inhibitor cell apoptosis, independent of their effect on HIV replication (zidovudine and saquinivir), and the other two-drug regimen had no protease inhibitors (zidovudine and zalitabine) Two-hundred and eighty subjects completed 24 weeks oftreatment. Even though there was worse virologic control in In vitro anti-apoptotic properties of HIV protease
the two-drug, than the three-drug regimen group, the sub- jects in the two-drug group who received the HIV PI hadimproved CD4+ T cell counts, compared to the two-drug Due to findings that PI therapy reduced lymphocyte apopto- group that did not receive an HIV PI. Subsequent clinical tri- sis, our group proposed in 1998 that highly active antiretro- als confirmed that HIV protease inhibitors improved CD4+ viral therapy (HAART) might independently block cellu- T cell counts in HIV-infected individuals, independent of the lar apoptosis Subsequent investigations tested and con- viral suppression In a meta-analysis comparing anti- firmed this finding ex vivo and in vitro HIV PIs HIV drug regimens using a PI to those that switched the PI anti-apoptotic effects were investigated using ritonavir in to a non-nucleoside reverse transcriptase inhibitor (NNRTI), cultures of bone marrow cells from HIV-infected patients or PI-based therapy resulted in higher CD4+ T cell counts normal controls. Hematopoietic colony forming unit replica- The analysis required that both PI and non-PI based regi- tion, following addition of ritonavir, had 45% less apoptosis mens have complete HIV viral suppression. Therefore, the than untreated cultures. The authors also reported a decrease CD4+ T cell benefit with HIV protease inhibitor therapy ap- in caspase-1 expression after ritonavir treatment peared to be, in part, unrelated to the effect on HIV viral In additional experiments investigating the effects of HIV suppression. In a different trial comparing treatment with PIs on T cell death during HIV infection, CD4+ T cells HIV protease containing highly active antiretroviral therapy isolated from healthy uninfected individuals had increased (HAART) to protease inhibitor therapy alone, there was less Fas expression and Fas and anti-CD3-induced apoptosis virologic suppression of the subjects on PI mono-therapy.
when incubated with HIV virions. Inducible Fas expres- However, there was no decrease in CD4+ T cell counts over sion and apoptosis were abrogated when the cells were pre- one year, even in the group who received PI mono-therapy incubated with the HIV PI, saquinavir These findings and virologically failed Additionally, in a comparison be- were supported by subsequent investigations which reported tween PI-based anti-HIV therapy to NNRTI-based anti-HIV that saquinavir and ritonavir inhibited TNF-mediated U937 therapy, there was equal viral suppression in both groups; cell apoptosis in a dose-dependent fashion with 38–60% re- however, PI-based regimens had a greater increase in CD4+ duction in apoptosis in PI treated cells During HIV infection, the HIV envelope gp120 binds to the CD4 and In the absence of effective antiretroviral therapy, HIV- CXCR4 receptors on the surface of the CD4+ T cell and infected subjects have considerable lymphocyte apoptosis in signals the cell to undergo apoptosis. This bystander death lymphoid tissue. The lymphoid tissue apoptosis is reduced is one of the ways that HIV depletes the immune system.

Apoptosis (2007) 12:969–977 Matarrese et al. treated human CD4+ T cells with HIV were expanded in a study that demonstrated that caspases-3, gp120 to make the cell sensitive to Fas-mediated apopto- -6, and -8 activity was not inhibited by indinavir in U937 cells sis, which resulted in mitochondrial changes and apoptosis at drug concentrations that effectively inhibited U937 apop- after Fas exposure. When the CD4+ T cells were pretreated tosis Nelfinavir did not block activation of caspases-1, with a PI before HIV gp120, mitochondrial depolorization -3, -4, -5, -9, and -8 in lysates of Jurkat T cells undergoing was blocked in a whole cell or cell-free system, or isolated Fas-mediated apoptosis mitochondria Taken together, this data indicates that In addition to caspases, the calpain proteases have been patients who received HIV protease inhibitors had improved considered as a possible site for HIV PIs to mediate apopto- CD4+ T cell counts independent of the state of HIV viral sis. Calpains are Ca+-dependent cysteine proteases reported replication, and in vitro work confirmed that HIV PIs can to be involved in several models of apoptosis, including U937 inhibit T cell apoptosis, specifically that induced by HIV.
cells, but are not absolutely required for apoptosis Be-cause HIV PIs are designed to inhibit the HIV cysteine pro-tease, they may influence cellular apoptosis by blocking cal- Mechanism of HIV protease inhibitors apoptosis
pain activation and function. Ghibelli et al. demonstrated in a U937 model of apoptosis that indinavir and ritonavir di-rectly inhibit apoptosis in cell systems where calpains are The mechanism of HIV protease inhibitor-mediated apopto- activated and block m-calpain activation Other inves- sis inhibition is being actively investigated (Table HIV PIs tigators have demonstrated that ritonavir competitively in- inhibit the proteolytic activity of HIV viral protease, there- hibited activity of both m- and -µ calpain isoforms in PC12 fore, it has been postulated that they might have a similar cells Other investigators could not confirm this ob- effect on other cellular proteases. Together with the obser- servation. These authors postulate that concentrations of ri- vations that HIV PIs block Fas-mediated apoptosis, many tonavir close to the maximum solubility of the drug, which investigators hypothesize that HIV PIs may inhibit the cas- was used in previous reports, may have artificially altered pase family members and block apoptosis. Although this is the results A calpain inhibitor could have significant an attractive model, caspases are cystine proteases and HIV therapeutic implications beyond HIV apoptosis, including protease is an aspartyle protease. Several groups have inves- neurodegenerative diseases; however, the evidence remains tigated the direct effect of HIV PIs on intracellular caspase unclear whether HIV PIs have a significant effect on calpain activity. When recombinant caspases-1, -3, -6, -7, or -8 were incubated with a fluorogenic tetrapeptide substrate for each An alternative model suggests that HIV protease in- caspase in the presence of absence of nelfinavir, the HIV hibitors alter the expression of apoptotic regulatory pro- PI inhibited HIV protease cleavage of gag/pol, but did not teins. Although early studies reported a change in Fas ex- inhibit the activity of any of the caspases These results pression after PI treatment, subsequent work did not show a Possible methods for HIV protease inhibitor regulating apoptosis Theoretical site of action HIV PIs block Fas and TNF mediated apoptosisHIV PIs do not block caspase-1, -3, -4, -5, -6, -7, -8, or -9 activity in HIV PIs inhibit apoptosis in cell systems where calpains are activatedHIV PIs blocked m-calpain activation in U937 cellsHIV PIs inhibited activity of both m- and -µ calpain isoforms in PC12 HIV PI did not inhibit m- or µ-calpain hydrolysis or activation at lower, Apoptosis regulatory proteins No change in Fas protein levels after HIV PI treatmentNo change in RNA levels of Fas, Fas L, and TNF after HIV PI treatmentNo change in Bcl-2, Bax, and Bcl-XL after HIV PI treatment Mitochondrial transmembrane potential HIV PIs maintain mitochondrial membrane integrity after apoptosis HIV PIs prevent cytochrome c release from mitochondria after apoptosis stimuli ANT (adenine nucleotide translocator) necessary for HIV PI to block mitochondria mediated apoptosis

Apoptosis (2007) 12:969–977 change in Fas levels with HIV PI therapy Bone release a fluorescent dye after pore opening, did not result marrow progenitor cells from HIV-infected individuals in- in pore opening when pretreated with nelfinavir before the cubated with ritonavir and indinavir showed no change in RNA levels of Fas, or Fas L. Two reports have specifically In summary, there are several theories regarding the mech- investigated the levels of intracellular apoptotic regulatory anism by which HIV PIs inhibit cellular apoptosis. The most proteins with and without HIV PI treatment. Protein levels data suggest that HIV PIs block apoptosis by maintaining of Bcl2, Bax, and Bcl-XL were evaluated by flow cytom- mitochondrial integrity, likely by HIV PIs preventing pore etry, and were unchanged after PI administration function of the adenine nucleotide translocator subunit of the Therefore, HIV PIs can block Fas- and TNF-mediated apop- mitochondrial permeability transition pore complex (Fig. tosis; this effect does not appear to be due to changes inintra- or extracellular apoptotic regulatory protein levels HIV protease inhibitor anti-apoptotic properties during
HIV PIs may also block apoptosis at the level of the non-HIV disease states
mitochondria by disrupting the transmitochondrial mem-brane potential. The mitochondrial transmembrane potential The novel anti-apoptotic properties of HIV PIs are being occurs from an asymmetric distribution of ions on both sides evaluated in preclinical studies as a potential therapy for dis- of the inner mitochondrial membrane that is maintained ease states associated with increased levels of apoptosis such by the mitochrondrial permeability transition pore complex as sepsis, the leading cause of death in critically ill patients.
(PTPC). After an apoptotic signal, the PTPC opens, The original description of in vivo use of HIV PIs for non- disrupts the membrane potential, and releases apoptogenic HIV related disease was in a mouse sepsis model. Sepsis is factors, including cytochrome c and procaspase-9. Thus, the the leading cause of death in critically ill patients. Despite mitochondria serves as a regulatory checkpoint of apoptotic advances in supportive care, mortality from sepsis remains signaling. Many regulatory proteins, including Bcl2 family high. Animal studies demonstrate that sepsis results in ex- members and IAPs, interact at the level of the mitochondria tensive lymphocyte apoptosis, as well as intestinal epithelial to alter apoptosis. In the first report of the effects of HIV cell apoptosis These findings have been confirmed PI on mitochondrial integrity, Jurkat cell Fas-mediated during autopsy studies in humans who died of sepsis apoptosis was inhibited with 10 µM of nelfinavir, a dose In a mouse model of sepsis, created by cecal ligation that would simulate physiologic levels if taken clinically.
and perforation, mice pretreated with HIV PIs had improved Nelfinavir treated cells maintained intact mitochondrial survival and reduced lymphocyte apoptosis The HIV transmembrane potential, as determined by DioC6 staining, PI treated mice had an increase in the Th1 cytokine TNFα a lipophilic dye that stains the mitochondria The and a reduction in the TH2 cytokines IL-6 and IL-10. It ap- authors also reported that 10 µM of nelfinavir inhibited cy- pears the beneficial effect of PI treatment was due to reduced tochrome c release during Fas-induced apoptosis. The HIV lymphocyte apoptosis because lymphocyte deficient Rag 1 accessory molecule, Vpr, caused PTPC opening and loss of –/– mice had no benefit from HIV PI treatment. Follow-up transmitochondrial potential when added directly to mito- work by Weaver et al. investigated the effect of HIV PIs chondria. Nelfinavir pretreatment of Jurkat cells prevented during Staphylococcal enterotoxin B+DGal-induced shock.
Vpr-induced DioC6 release from the mitochondria and There was a 60% improvement in 24-h survival in mice pre- cell death. Other groups have confirmed that HIV protease treated with HIV PIs than those treated with vehicle control.
inhibitors block mitochondrial transmembrane potential loss In addition, the authors also demonstrated that HIV PI pre- in multiple models of apoptosis In a recent treatment reduced mouse death from Fas-induced fatal hep- report, Weaver et al. investigated how HIV PIs influence atitis and middle cerebral artery occlusion-induced stroke mitochondrial integrity by using yeast models. Wild type or yeast deficient in voltage-dependent anion channel (VDAC)or adenine nucleotide translocator (ANT) isoforms, twocomponents of the PTPC, were treated with Vpr or H2O2 HIV protease inhibitors and stroke
which induce mitochondrial apoptosis. Apoptosis onlyoccurred after Vpr or H2O2 treatment when ANT was Cerebral ischemia or stroke occurs when blood flow (and present. Furthermore, when Jurkat cells were pretreated thus oxygen) to the brain is reduced through hemorrhage with nelfinavir and then an agonist for VDAC, there was or clot-induced occlusion of blood vessels. The only ther- no inhibition of mitochondrial potential loss or apoptosis.
apy with proven clinical benefit is thrombolysis requiring However, 10 µM of nelfinavir blocked ANT agonist-induced administration of tissue plasminogen activator within 3 h of mitochondrial transmembrane potential loss and apoptosis the onset of ischemic attack and/or oral aspirin within the first Lastly, proteoliposomes reconstituted with ANT, which 48 h after stroke onset Although the pathophysiology

Apoptosis (2007) 12:969–977 Fig. 1 HIV protease inhibitors block cellular apoptosis at the level of the adenine nucleotide translocase (ANT) in the mitochondrial pore complex.
The figure has been adapted from the Mitosciences Inc. website
of stroke damage extends well beyond this time frame, no Na+, Cl−, and Ca2+ ions. Neuronal depolarization resulting clinical intervention capable of targeting discrete molecular from a loss of calcium homeostasis is further exacerbated mechanisms of cell death has been shown to effectively pro- by the sustained release of glutamate and reduction in ex- tect neurons from subsequent neuronal loss. Elucidation of tracellular pH leading to excitotoxicity. Production of free the complex and multiple cell death pathways initiated by fatty acids and oxyradicals are elevated, triggering oxida- ischemia has made it increasingly apparent that a broader tive remodeling of membrane lipids, impaired glial home- therapeutic approach is required These findings are ostatic functions and enhanced inflammatory cell activation supported by correlative clinical evidence of neuroprotective activity in other central nervous system disorders following Studies using primary hippocampal neurons indicate that treatment with PIs HIV PIs can protect neurons from cell death triggered by Stroke elicits rapid necrotic and excitotoxic mechanisms membrane lipid remodeling associated with oxidative stress.
as well as delayed apoptotic-like responses. Cell death is Ritonavir has been shown to inhibit neuronal injury triggered initiated once core tissue is deprived of oxygen-rich blood.
in vitro by 4-hydroxynonenal, a lipid-soluble aldehydic prod- Immediately following vessel occlusion, cell viability is lost, uct of membrane peroxidation that impairs Na+, K+, and - in part, by a reduction in ATP levels resulting in an efflux of ATPase activity In vivo, administration of nelfinavir and K+ ions from compromised neurons and glia and an influx of ritonavir prior to ischemic insult effectively reduces infarct

Apoptosis (2007) 12:969–977 size in mice following middle cerebral artery occlusion, re-sulting in functional recovery of ischemic neurons Mechanistic assessment suggests that HIV PIs act to re- duce delayed neuronal ischemic death triggered also throughmitochondrial-dependent pathways. Neurons located at theperiphery of the necrotic core (dubbed the penumbra) arespared acute ischemic injury. Damage is not evident untilhours, days, and weeks following reperfusion when cells be-gin to exhibit many of the morphological and biochemicalcharacteristics of apoptosis Death is likely triggeredduring ischemia/reperfusion by downstream release of in-ternal calcium stores from endoplasmic reticulum and mito-chondria Additional death pathways are subsequentlyinitiated by a complex cross-talk between extrinsic deathreceptor mediated-induction and intrinsic mitochondrial-dependent pathways. Briefly, extrinsic induction involvesbinding of apoptogens, such as Fas ligand and tumor necro-sis factor α (TNFα), to death domain-containing receptorsinducing oligomerization and recruitment of the adapter pro-teins FADD and TRADD Complex formation betweenadapter proteins, receptor death domains, and procaspasesaccelerates the autocatalytic activation of procaspase-8, -10,and -2. Once cleaved, these initiator caspases can cleave andactivate executioner caspases-3, -6, and -7 responsible for theregulated disassembly of cellular proteins characteristic ofapoptosis Intrinsic cell death is dictated by mitochon-drial function. Release of reactive oxygen species followingischemic reperfusion triggers release of cytochrome c andATP from compromised mitochondria into the cytosol, oftenwith concomitant loss of loss of ym. Activation of caspase9 occurs with formation of the apoptosome composed ofAPAF-1, cytochrome c, ATP, and procaspase 9 resulting in Effects of nelfinavir and ritonavir on ischemic hippocampal downstream activation of caspase-3, -6, -7 damage induced by two vessel carotid occlusion. Mice subjected to HIV PIs act at multiple mitochondrial branch points of two vessel occlusion (A) were compared to animals that received three these overlapping apoptotic cascades to reduce delayed neu- oral gavages of either nelfinavir or ritonavir 8 h apart starting 24 h ronal death following ischemic insult. Pretreatment with nel- before (B), 1 h after (C), or 6 h after (D) carotid occlusion. Arrowsindicate areas of ischemic damage. Viable cell number in the CA1 was finavir prior to apoptotic challenge inhibits the pore function counted 48 h after ischemic onset in hemotoxylin and eosin-stained of the adenine nucleotide transporter, thereby reducing the sections (E). Data represent the average number of viable cells in loss of loss of y both hippocampi and are expressed per animal. The mean number of m, apoptosome formation, and downstream viable cells per condition ± standard error of measurement is indi- caspase activation Because HIV PIs inhibit the down- cated. Neurodegeneration was completely attenuated by pre-treatment stream mitochondrial events elicited by caspase 8-mediaed with PIs and partially attenuated by administration of PIs following cleavage of Bid, downstream events of the extrinsic pathway carotid occlusion. Scale bars, 100 µm are also reduced Recent unpublished data from our laboratories indicate that this protection may also be of potential clinical benefit mals received nelfinavir boosted by ritonavir prior to or im- even if administered after the ischemic event—a key require- mediately after 2VO surgery (Fig. Neuroprotection was ment for successful adjuvant therapies. Loss of CA1 pyra- still evident, albeit at reduced efficacy, when nelfinavir and midal neurons in the hippocampus is a hallmark of global ritonavir were administered up to 6 h after two-vessel carotid ischemia. Ischemic hippocampal injury can be experimen- occlusion (Fig. suggesting a wider therapeutic window tally designed by the two-vessel occlusion rodent model of than previously anticipated global ischemia in which both internal carotid arteries are While promising, mechanistic assessment of HIV PIs transiently ligated. As demonstrated in Fig. we observed in stroke management is still in early days. Although significant sparing of CA1 pyramidal neurons whether ani- HIV PIs appear to inhibit apoptosis in multiple organs at Apoptosis (2007) 12:969–977 concentrations comparable to that seen in the plasma follow- drug may interfere with cellular activation signals that result ing clinical application (reviewed in these same com- in transformation and protect the tumor cell from apopto- pounds trigger cell death at higher concentrations sis. High concentrations of ritonavir (10–50 µM) inhibit the Moreover, chronic administration, as part of HIV treatment, proliferation of murine and human tumor cell lines. DNA has been associated with an enhanced risk of ischemia in laddering demonstrated that 15–30 µM of ritonavir induced both central and peripheral tissue. Chronic HIV PI treatment apoptosis in the same cell lines Of note, the concen- can elicit hyperlipidemia, accelerating atherosclerosis and trations of ritonavir used were over 15 times what most represent a potential increased risk for myocardial infarction adults achieve at FDA-approved doses and at which the As such, it is unlikely that HIV PIs can be used drug blocks apoptosis. This led to further work developing prophylactically to prevent stroke damage in susceptible in- the pro-apoptotic effects of HIV PIs. Adult T-cell leukemia dividuals. However, the promise of transient administration (ATL) is an aggressive malignancy associated with human is promising. The impact of acute administration of HIV T-cell leukemia virus (HTLV) and is very resistant to con- PIs following stroke requires a thorough evaluation of the ventional chemotherapy. When ATL cells were incubated therapeutic window open to mitochondrial manipulation and with 20–40 µM of ritonavir, there was a five-fold increase is essential to validate the promise of HIV PIs as a poten- in spontaneous apoptosis, resulting in a similar decrease in tial adjuvant strategy to promote neuronal survival following tumor cell survival In ATL cell lines and primary ATL cells 40 µM of ritonavir inhibited transcriptional activationof NF-κB. In addition, HIV PIs inhibited the expression ofthe targets of NF-κB, Bcl-XL, survivin, c-Myc, and cyclin D2 HIV protease inhibitors and pancreatitis
There is also evidence that HIV protease inhibitors are Accelerated apoptosis also contributes to cellular injury dur- pro-apoptotic in models of solid tumors. Freshly isolated ing acute pancreatitis. Our group investigated whether treat- multiple myeloma cells from patients under went apopto- ment with the HIV protease inhibitors nelfinavir/ritonavir sis after incubation with 40–50 µM of ritonavir, saquinavir, would reduce the severity of pancreatitis using a mouse and nelfinavir. This was associated with a decrease in caerulein-induced pancreatitis model. Ritonavir was used to the anti-apoptotic protein Mcl-1, and blocked IL-6 phos- increase nelfinavir drug levels to a therapeutic level in the phorylation of ERK 1/2 and STAT 3 Different tu- mice. Mice treated with nelfinavir/ritonavir before caerulein mor types may have different mechanisms by which HIV induced pancreatitis had reduced serum amylase levels and PIs inhibit tumor growth and promote apoptosis. Riton- less acinar injury of the pancreas on histologic review, com- avir used at 20 µM in solid and hematologic tumor models pared to mice pretreated with vehicle control.
caused an increase in the cellular concentrations of the anti-proliferative and pro-apoptotic proteasome substrate cdk in-hibitor, p21. This was associated with a block in prote- HIV protease inhibitors and T cell production
olytic degradation consistent with an earlier reportthat HIV PI impact proteasome activity Accumula- The anti-apoptotic effects of HIV protease inhibitors may tion of intracellular p21 resulted in cell-cycle arrest in G1 have indirect beneficial clinical effects as well. In a recent re- phase and subsequent apoptosis in ritonavir treated tumor port by Graham et al., five out of seven HIV-negative patients treated with an HIV protease inhibitor containing HAART HIV protease inhibitors are well-tolerated drugs with en- regimen for a needle-stick exposure experienced a 3-log in- ticing possibilities for future use to alter apoptosis in many crease in thymic-derived na¨ıve T cells in the peripheral blood.
human disease states. Clinical trials of HIV PIs in disease The increase in na¨ıve T cells, as determined by T cell recep- states other than HIV are already underway investigating the tor recombination excision circle levels (TREC), occurred in vivo effects on human cellular apoptosis (NCT00346619 after four weeks of therapy, suggesting, in yet another set- and NCT00233948). Furthermore, now that the target for ting, that HIV PI can have beneficial immunemodulatory how HIV PIs block cellular apoptosis has been identi- fied as the mitochondrial pore protein ANT, physiochemi-cal optimization can be performed. Lastly, the mechanismby which high concentrations of HIV PIs induce apopto- Paradoxical pro-apoptotic effect of HIV protease
sis in transformed tumor models needs to be further clari- fied. Studies are needed to determine whether the HIV PIsalter proteins that modulate cellular proliferation or apop- Under certain conditions HIV PIs may be pro-apoptotic as tosis, and whether there is a threshold to the pro-apoptotic well. At increased doses of HIV PI, it is possible that the Apoptosis (2007) 12:969–977 SRV is supported by a grant from the Mayo 16. Matarrese P, Tinari A, Gambardella L et al (2005) HIV pro- Program in Translational Immunovirology and Biodefense and a Robert tease inhibitors prevent mitochondrial hyperpolarization and re- and Arlene Kogod Program on Aging Award from the Mayo Foun- dox imbalance and decrease endogenous uncoupler protein-2 ex- dation. ADB is supported by NIH grants RO1-AI62261 and RO1- pression in gp 120-activated human T lymphocytes. Antivir Ther AI40384, and a Burroughs Wellcome Award ID#1005160.
10(Suppl 2):M29–M45 17. Phenix BN, Lum JJ, Nie Z, Sanchez-Dardon J, Badley AD. (2001) Antiapoptotic mechanism of HIV protease inhibitors: preventing mitochondrial transmembrane potential loss. Blood 98(4):1078–1085 18. Ghibelli L, Mengoni F, Lichtner M et al (2003) Anti-apoptotic 1. Collier AC, Coombs RW, Schoenfeld DA et al (1996) Treatment of effect of HIV protease inhibitors via direct inhibition of calpain.
human immunodeficiency virus infection with saquinavir, zidovu- Biochem Pharmacol 66(8):1505–1512 dine, and zalcitabine: AIDS Clinical Trials Group. N Engl J Med 19. Chavan S, Kodoth S, Pahwa R, Pahwa S (2001) The HIV pro- tease inhibitor Indinavir inhibits cell-cycle progression in vitro in 2. Kravcik S, Magill A, Sanghvi B et al (2001) Comparative CD4 lymphocytes of HIV-infected and uninfected individuals. Blood T-cell responses of reverse transcriptase inhibitor therapy with or without nelfinavir matched for viral exposure. HIV Clin Trials 20. Spinedi A, Oliverio S, Di Sano F, Piacentini M (1998) Calpain in- volvement in calphostin C-induced apoptosis. Biochem Pharmacol 3. Deeks SG, Grant RM. (1999) Sustained CD4 responses after vi- rological failure of protease inhibitor-containing therapy. Antivir 21. Wan W, DePetrillo PB (2002) Ritonavir inhibition of calcium- Ther 4(Suppl 3):7–11 activated neutral proteases. Biochem Pharmacol 63(8):1481–1484 4. Albrecht MA, Bosch RJ, Hammer SM et al (2001) Nelfinavir, 22. Cuerrier D, Nie Z, Badley AD, Davies PL (2005) Ritonavir efavirenz, or both after the failure of nucleoside treatment of HIV does not inhibit calpain in vitro. Biochem Biophys Res Commun infection. N Engl J Med 345(6):398–407 5. Staszewski S, Morales-Ramirez J, Tashima KT et al (1999) 23. Sloand EM, Kumar PN, Kim S, Chaudhuri A, Weichold FF, Young Efavirenz plus zidovudine and lamivudine, efavirenz plus indi- NS (1999) Human immunodeficiency virus type 1 protease in- navir, and indinavir plus zidovudine and lamivudine in the treat- hibitor modulates activation of peripheral blood CD4(+) T cells ment of HIV-1 infection in adults. Study 006 Team. N Engl J Med and decreases their susceptibility to apoptosis in vitro and in vivo.
Blood 94(3):1021–1027 6. Owen C, Kazim F, Badley AD (2004) Effect on CD4 T-cell count 24. Lu W, Andrieu JM (2000) HIV protease inhibitors restore of replacing protease inhibitors in patients with successful HIV impaired T-cell proliferative response in vivo and in vitro: suppression: a meta-analysis. Aids 18(4):693–695 a viral-suppression-independent mechanism. Blood 96(1):250– 7. Arribas JR, Pulido F, Delgado R et al (2005) Lopinavir/ritonavir as single-drug therapy for maintenance of HIV-1 viral suppression: 25. Isgro A, Aiuti A, Mezzaroma I et al (2005) HIV type 1 protease 48-week results of a randomized, controlled, open-label, proof-of- inhibitors enhance bone marrow progenitor cell activity in nor- concept pilot clinical trial (OK Study). J Acquir Immune Defic mal subjects and in HIV type 1-infected patients. AIDS Res Hum Syndr 40(3):280–287 8. Fethi T, Asma J, Amine SM et al (2005) Effects on immunological 26. Matarrese P, Gambardella L, Cassone A, Vella S, Cauda R, Mal- and virological outcome of patients using one protease inhibitor orni W (2003) Mitochondrial membrane hyperpolarization hijacks or one non-nucleoside reverse transcriptase inhibitor in a triple activated T lymphocytes toward the apoptotic-prone phenotype: antiretroviral therapy: normal clinical practice versus clinical trial homeostatic mechanisms of HIV protease inhibitors. J Immunol findings. Curr HIV Res 3(3):271–276 9. Badley AD, Dockrell DH, Algeciras A et al (1998) in vivo analysis 27. Weichold FF, Bryant JL, Pati S, Barabitskaya O, Gallo RC, Reitz Jr of Fas/FasL interactions in HIV-infected patients. J Clin Invest MS (1999) HIV-1 protease inhibitor ritonavir modulates suscepti- bility to apoptosis of uninfected T cells. J Hum Virol 2(5):261–269 10. Landay AL, Spritzler J, Kessler H et al (2003) Immune recon- stitution is comparable in antiretroviral-naive subjects after 1 Mitochondrion-mediated apoptosis in HIV-1 infection. Trends year of successful therapy with a nucleoside reverse-transcriptase Pharmacol Sci 24(6):298–305 inhibitor- or protease inhibitor-containing antiretroviral regimen. J 29. Garg H, Blumenthal R (2006) HIV gp41-induced apoptosis is Infect Dis 188(10):1444–1454 mediated by caspase-3-dependent mitochondrial depolarization, 11. Badley AD, Parato K, Cameron DW et al (1999) Dynamic correla- which is inhibited by HIV protease inhibitor nelfinavir. J Leukoc tion of apoptosis and immune activation during treatment of HIV Biol 79(2):351–362 infection. Cell Death Differ 6(5):420–432 30. Miro O, Villarroya J, Garrabou G et al (2005) in vivo effects of 12. Phenix BN, Angel JB, Mandy F et al (2000) Decreased HIV- highly active antiretroviral therapies containing the protease in- associated T cell apoptosis by HIV protease inhibitors. AIDS Res hibitor nelfinavir on mitochondrially driven apoptosis. Antivir Ther Hum Retroviruses 16(6):559–567 13. Sloand EM, Maciejewski J, Kumar P, Kim S, Chaudhuri A, Young 31. Weaver JG, Tarze A, Moffat TC et al (2005) Inhibition of ade- N. (2000) Protease inhibitors stimulate hematopoiesis and de- nine nucleotide translocator pore function and protection against crease apoptosis and ICE expression in CD34(+) cells. Blood apoptosis in vivo by an HIV protease inhibitor. J Clin Invest 14. Estaquier J, Lelievre JD, Petit F et al (2002) Effects of antiretroviral 32. Ayala A, Herdon CD, Lehman DL, Ayala CA, Chaudry IH (1996) drugs on human immunodeficiency virus type 1-induced CD4(+) Differential induction of apoptosis in lymphoid tissues during sep- T-cell death. J Virol 76(12):5966–5973 sis: variation in onset, frequency, and the nature of the mediators.
15. Wolf T, Findhammer S, Nolte B, Helm EB, Brodt HR. (2003) Blood 87(10):4261–4275 Inhibition of TNF-alpha mediated cell death by HIV-1 specific 33. Hotchkiss RS, Swanson PE, Cobb JP, Jacobson A, Buchman TG, protease inhibitors. Eur J Med Res 8(1):17–24 Karl IE (1997) Apoptosis in lymphoid and parenchymal cells dur- Apoptosis (2007) 12:969–977 ing sepsis: findings in normal and T- and B-cell-deficient mice.
49. Pajonk F, Himmelsbach J, Riess K, Sommer A, McBride WH Crit Care Med 25(8):1298–1307 (2002) The human immunodeficiency virus (HIV)-1 protease in- 34. Husain KD, Coopersmith CM (2003) Role of intestinal epithelial hibitor saquinavir inhibits proteasome function and causes apop- apoptosis in survival. Curr Opin Crit Care 9(2):159–163 tosis and radiosensitization in non-HIV-associated human cancer 35. Coopersmith CM, Chang KC, Swanson PE et al (2002) Overex- cells. Cancer Res 62(18):5230–5235 pression of Bcl-2 in the intestinal epithelium improves survival in 50. Zhong DS, Lu XH, Conklin BS et al (2002) HIV protease in- septic mice. Crit Care Med 30(1):195–201 hibitor ritonavir induces cytotoxicity of human endothelial cells.
36. Weaver JG, Rouse MS, Steckelberg JM, Badley AD (2004) Im- Arterioscler Thromb Vasc Biol 22(10):1560–1566 proved survival in experimental sepsis with an orally administered 51. Bode H, Lenzner L, Kraemer OH et al (2005) The HIV protease inhibitor of apoptosis. Faseb J 18(11):1185–1191 inhibitors saquinavir, ritonavir, and nelfinavir induce apoptosis and 37. Ly JV, Zavala JA, Donnan GA (2006) Neuroprotection and throm- decrease barrier function in human intestinal epithelial cells. An- bolysis: combination therapy in acute ischaemic stroke. Expert tivir Ther 10(5):645–655 Opin Pharmacother 7(12):1571–1581 52. Penzak SR, Chuck SK (2002) Management of protease inhibitor- 38. Lee JM, Zipfel GJ, Choi DW (1999) The changing landscape of associated hyperlipidemia. Am J Cardiovasc Drugs 2(2):91– ischaemic brain injury mechanisms. Nature 399(6738 Suppl):A7– 53. Lai S, Lai H, Celentano DD et al (2003) Factors associated with 39. Reed JC (2001) Apoptosis-regulating proteins as targets for drug accelerated atherosclerosis in HIV-1-infected persons treated with discovery. Trends Mol Med 7(7):314–319 protease inhibitors. AIDS Patient Care STDS 17(5):211–219 40. Lo EH, Moskowitz MA, Jacobs TP (2005) Exciting, radical, sui- 54. Cohen CJ (2005) Ritonavir-boosted protease inhibitors, Part 2: cidal: how brain cells die after stroke. Stroke 36(2):189–192 cardiac implications of lipid alterations. AIDS Read 15(10):528– 41. Rosenfeldt V, Valerius NH, Paerregaard A (2000) Regression of HIV-associated progressive encephalopathy of childhood during 55. Graham DB, Bell MP, Huntoon CJ et al (2005) Increased thymic HAART. Scand J Infect Dis 32(5):571–574 output in HIV-negative patients after antiretroviral therapy. Aids 42. MacGowan DJ, Scelsa SN, Waldron M (2001) An ALS-like syn- drome with new HIV infection and complete response to antiretro- 56. Gaedicke S, Firat-Geier E, Constantiniu O et al (2002) Antitumor viral therapy. Neurology 57(6):1094–1097 effect of the human immunodeficiency virus protease inhibitor 43. Lopez-Neblina F, Toledo AH, Toledo-Pereyra LH (2005) Molecu- ritonavir: induction of tumor-cell apoptosis associated with per- lar biology of apoptosis in ischemia and reperfusion. J Invest Surg turbation of proteasomal proteolysis. Cancer Res 62(23):6901– 44. MacDonald JF, Xiong ZG, Jackson MF (2006) Paradox of Ca2+ 57. Dewan MZ, Uchihara JN, Terashima K et al (2006) Efficient inter- signaling, cell death and stroke. Trends Neurosci 29(2):75–81 vention of growth and infiltration of primary adult T-cell leukemia 45. Wan W, DePetrillo PB (2002) Ritonavir protects hippocampal neu- cells by an HIV protease inhibitor, ritonavir. Blood 107(2):716– rons against oxidative stress-induced apoptosis. Neurotoxicology 58. Ikezoe T, Saito T, Bandobashi K, Yang Y, Koeffler HP, Taguchi H 46. Ferrer I, Planas AM (2003) Signaling of cell death and cell survival (2004) HIV-1 protease inhibitor induces growth arrest and apop- following focal cerebral ischemia: life and death struggle in the tosis of human multiple myeloma cells via inactivation of signal penumbra. J Neuropathol Exp Neurol 62(4):329–339 transducer and activator of transcription 3 and extracellular signal- 47. Zheng Z, Zhao H, Steinberg GK, Yenari MA (2003) Cellular and regulated kinase 1/2. Mol Cancer Ther 3(4):473–479 molecular events underlying ischemia-induced neuronal apoptosis.
59. Andre P, Groettrup M, Klenerman P et al (1998) An inhibitor of Drug News Perspect 16(8):497–503 HIV-1 protease modulates proteasome activity, antigen presenta- 48. Phenix BN, Cooper C, Owen C, Badley AD (2002) Modulation of tion, and T cell responses. Proc Natl Acad Sci USA 95(22):13120– apoptosis by HIV protease inhibitors. Apoptosis 7(4):295–312

Source: http://neurolipidomics.org/NRL%20publications/2007Valhakis.pdf