Assessing the degree of hepatic ischemia-reperfusion injury using PBPK modeling of sodium fluorescein disposition in ex vivo machine-perfused livers.

Ischemia-reperfusion injury (IRI) is an intrinsic risk associated with liver transplantation. Ex vivo hepatic machine perfusion (MP) is an emerging organ preservation technique that can mitigate IRI, especially in livers subjected to prolonged warm ischemia time (WIT). However, a method to quantify the biological response to WIT during MP has not been established. Previous studies used physiologically-based pharmacokinetic (PBPK) modeling to demonstrate that a decrease in hepatic transport and biliary excretion of the tracer molecule sodium fluorescein (SF) could correlate with increasing WIT in situ. Furthermore, these studies proposed intracellular sequestration of the hepatocyte canalicular membrane transporter multi-drug resistance-associated protein 2 (MRP2) leading to decreased MRP2 activity (maximal transport velocity; Vmax) as the potential mechanism for decreased biliary SF excretion. We adapted an extant PBPK model to account for ex vivo hepatic MP and fit a 6-parameter version of this model to control time course measurements of SF in MP perfusate and bile. We then identified parameters whose values were likely insensitive to changes in WIT and fixed them to generate a reduced model with only 3 unknown parameters. Finally, we fit the reduced model to each individual biological replicate SF time course with differing WIT and found the mean estimated value for each parameter and compared them using a one-way ANOVA. We demonstrated that there was a significant decrease in the estimated value of Vmax for MRP2 at 30 min WIT. These studies provide the foundation for future studies investigating real-time assessment of liver viability during ex vivo MP.

Fluorescein clearance kinetics in blood and bile indicates hepatic ischemia-reperfusion injury in rats.

Quantitative measurement of the degree of hepatic ischemia-reperfusion injury (IRI) is crucial for developing therapeutic strategies for its treatment. We hypothesized that clearance of fluorescent dye through bile metabolism may reflect the degree of hepatic IRI. In this study, we investigated sodium fluorescein clearance kinetics in blood and bile for quantifying the degree of hepatic IRI. Warm ischemia times (WITs) of 0, 30, or 60 min followed by 1 h or 4 h of reperfusion, were applied to the median and lateral lobes of the liver in Sprague-Dawley rats. Subsequently, 2 mg/kg of sodium fluorescein was injected intravenously, and blood and bile samples were collected over 60 min to measure fluorescence intensities. The bile-to-plasma fluorescence ratios demonstrated an inverse correlation with WIT and were distinctly lower in the 60-min WIT group than in the control or 30-min WIT groups. Bile-to-plasma fluorescence ratios displayed superior discriminability for short versus long WITs when measured 1 h after reperfusion versus 4 h. We conclude that the bile-to-blood ratio of fluorescence after sodium fluorescein injection has the potential to enable the quantification of hepatic IRI severity.NEW & NOTEWORTHY Previous attempts to use fluorophore clearance to test liver function have relied on a single source of data. However, the kinetics of substrate processing via bile metabolism include decreasing levels in blood and increasing levels in bile. Thus, we analyzed data from blood and bile to better reflect fluorescein clearance kinetics.

Analogs of the Heat Shock Protein 70 Inhibitor MKT-077 Suppress Medullary Thyroid Carcinoma Cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC.

Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells.

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels.

While cellular LC3B and SQSTM1 levels serve as key autophagy markers, their regulation by different signaling pathways requires better understanding. Here, we report the mechanisms by which the Raf/MEK/ERK pathway regulates cellular LC3B and SQSTM1 levels. In different cell types, ΔRaf-1:ER- or B-Raf(V600E)-mediated MEK/ERK activation increased LC3B-I, LC3B-II, and SQSTM1/p62 levels, which was accompanied by increased BiP/GRP78 expression. Use of the autophagy inhibitors chloroquine and bafilomycin A1, or RNA interference of ATG7, suggested that these increases in LC3B and SQSTM1 levels were in part attributed to altered autophagic flux. However, intriguingly, these increases were also attributed to their increased expression. Upon Raf/MEK/ERK activation, mRNA levels of LC3B and SQSTM1 were also increased, and subsequent luciferase reporter analyses suggested that SQSTM1 upregulation was mediated at transcription level. Under this condition, transcription of BiP/GRP78 was also increased, which was necessary for Raf/MEK/ERK to regulate LC3B at the protein, but not mRNA, level. This suggests that BiP has a role in regulating autophagy machinery when Raf/MEK/ERK is activated. In conclusion, these results suggest that, under a Raf/MEK/ERK-activated condition, the steady-state cellular levels of LC3B and SQSTM1 can also be determined by their altered expression wherein BiP is utilized as an effector of the signaling.

AKT upregulates B-Raf Ser445 phosphorylation and ERK1/2 activation in prostate cancer cells in response to androgen depletion.

Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation. Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (∆4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated B-Raf regulation was also detected in a panel of PCa lines derived from the cPten(-/-)L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells.

Physiologically-Based Pharmacokinetic Modeling of Blood Clearance of Liver Fluorescent Markers for the Assessment of the Degree of Hepatic Ischemia-Reperfusion Injury.

During liver transplantation, ischemia-reperfusion injury (IRI) is inevitable and decreases the overall success of the surgery. While guidelines exist, there is no reliable way to quantitatively assess the degree of IRI present in the liver. Our recent study has shown a correlation between the bile-to-plasma ratio of FDA-approved sodium fluorescein (SF) and the degree of hepatic IRI, presumably due to IRI-induced decrease in the activity of the hepatic multidrug resistance-associated protein 2 (MRP2); however, the contribution of SF blood clearance via the bile is still convoluted with other factors, such as renal clearance. In this work, we sought to computationally model SF blood clearance via the bile. First, we converted extant SF fluorescence data from rat whole blood, plasma, and bile to concentrations using calibration curves. Next, based on these SF concentration data, we generated a "liver-centric", physiologically-based pharmacokinetic (PBPK) model of SF liver uptake and clearance via the bile. Model simulations show that SF bile concentration is highly sensitive to change in the activity of hepatic MPR2. These simulations suggest that SF bile clearance along with the PBPK model can be used to quantify the effect of IRI on the activity of MRP2.Clinical Relevance- This study establishes the theory necessary to generate a model for predicting the degree of IRI during liver transplantation.

Prolonged warm ischemia time increases mitogen-activated protein kinase activity and decreases perfusate cytokine levels in ex vivo rat liver machine perfusion.

Introduction: Machine perfusion is increasingly being utilized in liver transplantation in lieu of traditional cold static organ preservation. Nevertheless, better understanding of the molecular mechanisms underlying the ischemia-reperfusion injury (IRI) during ex vivo perfusion is necessary to improve the viability of liver grafts after transplantation using machine perfusion technology. Since key cellular signaling pathways involved in hepatic IRI may allow a chance for designing a promising approach to improve the clinical outcomes from this technology, we determined how warm ischemia time (WIT) during procurement affects the activity of mitogen-activated protein kinase (MAPK) and perfusate concentration of cytokines in an ex vivo rat liver machine perfusion model. Methods: Male Sprague-Dawley rats underwent in situ hepatic ischemia with varying WIT (0, 10, 20, 30 min, n = 5 each), and subsequently 3 h of cold ischemia time and 2 h of machine perfusion prior to determining the degree of MAPK activation-phosphorylation and cytokine concentration in liver tissue and perfusates, respectively. Results: Our data revealed a strong correlation between incremental WIT and a series of liver injury markers, and that prolonged WIT increases ERK1/2 and p54 JNK phosphorylation during machine perfusion. Notably, specific cytokine levels (MCP-1, MIP-2, GRO/KC, IL-10, and IL-5) were inversely correlated with the phosphorylation levels of ERK1/2, p38 MAPK, and p46/p54 JNK. Discussion: These results suggest that MAPK activation, specifically ERK1/2 and p54 JNK phosphorylation, have potential as a biomarker for hepatic IRI pathophysiology during machine perfusion. Elucidation of their functional significance may lead to designing a novel strategy to increase the clinical benefit of machine perfusion.

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells.

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16(INK4A) and p21(CIP1), but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.

Mortalin depletion induces MEK/ERK-dependent and ANT/CypD-mediated death in vemurafenib-resistant B-RafV600E melanoma cells.

Therapy resistance to a selective B-Raf inhibitor (BRAFi) poses a challenge in treating patients with BRAF-mutant melanomas. Here, we report that RNA interference of mortalin (HSPA9/GRP75), a mitochondrial molecular chaperone often upregulated and mislocalized in melanoma, can effectively induce death of vemurafenib-resistant progenies of human B-RafV600E melanoma cell lines, A375 and Colo-829. Mortalin depletion induced death of vemurafenib-resistant cells at similar efficacy as observed in vemurafenib-naïve parental cells. This lethality was correlated with perturbed mitochondrial permeability and was attenuated by knockdown of adenine nucleotide translocase (ANT) and cyclophilin D (CypD), the key regulators of mitochondrial permeability. Chemical inhibition of MEK1/2 and ERK1/2 also suppressed mortalin depletion-induced death and mitochondrial permeability in these cells. These data suggest that mortalin and MEK/ERK regulate an ANT/CypD-associated mitochondrial death mechanism(s) in B-RafV600E melanoma cells and that this regulation is conserved even after these cells develop BRAFi resistance. We also show that doxycycline-induced mortalin depletion can effectively suppress the xenografts of vemurafenib-resistant A375 progeny in athymic nude mice. These findings suggest that mortalin has potential as a candidate therapeutic target for BRAFi-resistant BRAF-mutant tumors.

Noncatalytic function of ERK1/2 can promote Raf/MEK/ERK-mediated growth arrest signaling.

Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.

Mortalin/HSPA9 targeting selectively induces KRAS tumor cell death by perturbing mitochondrial membrane permeability.

The mitochondrial HSP70 chaperone mortalin (HSPA9/GRP75) is often upregulated and mislocalized in MEK/ERK-deregulated tumors. Here, we show that mortalin depletion can selectively induce death of immortalized normal fibroblasts IMR90E1A when combined with K-RasG12V expression, but not with wild-type K-Ras expression, and that K-RasG12V-driven MEK/ERK activity is necessary for this lethality. This cell death was attenuated by knockdown or inhibition of adenine nucleotide translocase (ANT), cyclophilin D (CypD), or mitochondrial Ca2+ uniporter (MCU), which implicates a mitochondria-originated death mechanism. Indeed, mortalin depletion increased mitochondrial membrane permeability and induced cell death in KRAS-mutated human pancreatic ductal adenocarcinoma (PDAC) and colon cancer lines, which were attenuated by knockdown or inhibition of ANT, CypD, or MCU, and occurred independently of TP53 and p21CIP1. Intriguingly, JG-98, an advanced MKT-077 derivative, phenocopied the lethal effects of mortalin depletion in K-RasG12V-expressing IMR90E1A and KRAS-mutated tumor cell lines in vitro. Moreover, JG-231, a JG-98 analog with improved microsomal stability effectively suppressed the xenograft of MIA PaCa-2, a K-RasG12C-expressing human PDAC line, in athymic nude mice. These data demonstrate that oncogenic KRAS activity sensitizes cells to the effects of mortalin depletion, suggesting that mortalin has potential as a selective therapeutic target for KRAS-mutated tumors.

Mortalin (HSPA9) facilitates BRAF-mutant tumor cell survival by suppressing ANT3-mediated mitochondrial membrane permeability.

Mortalin [also known as heat shock protein family A (HSP70) member 9 (HSPA9) or glucose-regulated protein 75 (GRP75)] is a mitochondrial molecular chaperone that is often up-regulated and mislocalized in tumors with abnormal activation of the kinases MEK and ERK. Here, we found that mortalin depletion was selectively lethal to tumor and immortalized normal cells expressing the mutant kinase B-RafV600E or the chimeric protein ΔRaf-1:ER and that MEK-ERK-sensitive regulation of the peptide-binding domain in mortalin was critical to cell survival or death. Proteomics screening identified adenine nucleotide translocase 3 (ANT3) as a previously unknown mortalin substrate and cell survival/death effector. Mechanistically, increased MEK-ERK signaling activity and mortalin function converged opposingly on the regulation of mitochondrial permeability. Specifically, whereas MEK-ERK activity increased mitochondrial permeability by promoting the interaction between ANT3 and the peptidyl-prolyl isomerase cyclophilin D (CypD), mortalin decreased mitochondrial permeability by inhibiting this interaction. Hence, mortalin depletion increased mitochondrial permeability in MEK-ERK-deregulated cells to an extent that triggered cell death. HSP70 inhibitor derivatives that effectively inhibited mortalin suppressed the proliferation of B-RafV600E tumor cells in culture and in vivo, including their B-Raf inhibitor-resistant progenies. These findings suggest that targeting mortalin has potential as a selective therapeutic strategy in B-Raf-mutant or MEK-ERK-driven tumors.

A glutaredoxin-fused thiol peroxidase acts as an important player in hydrogen peroxide detoxification in late-phased growth of Anabaena sp. PCC7120.

The Anabaena sp. genome contains an open reading frame with homology to a novel hybrid form of thiol peroxidase, fused with a glutaredoxin domain. The gene was expressed in Escherichia coli. The purified hybrid protein exerted the highest peroxidase activity toward H2O2 using an electron from a reduced form of glutathione. The calculated kcat and kcat/K(m) values for H2O2 are 48.2 s(-1) and 3.29 x 10(6) M(-1) s(-1), respectively. Immunoblot analyses of the heterocystic proteins showed that the level of the protein in the heterocyst is comparable to that in the vegetative cell. All oxidants tested significantly elevated the mRNA and protein levels. The transcript slightly increased during the exponential growth phase, following which it increased steeply. Also, the levels of transcript were significantly increased in response to N2 starvation, carbon starvation, and light elimination. Taken together, the present data reveal for the first time that the glutathione-dependent thiol peroxidase is an adaptive strategy in Anabaena sp. that efficiently combats H2O2 that are produced during later phase of vegetative and heterocystic growth.

A cellular threshold for active ERK1/2 levels determines Raf/MEK/ERK-mediated growth arrest versus death responses.

In addition to its conventional role for cell proliferation and survival, the Raf/MEK/Extracellular signal-regulated kinase (ERK) pathway can also induce growth arrest and death responses, if aberrantly activated. Here, we determined a molecular basis of ERK1/2 signaling that underlies these growth inhibitory physiological outputs. We found that overexpression of ERK1 or ERK2 switches ΔRaf-1:ER-induced growth arrest responses to caspase-dependent apoptotic death responses in different cell types. These death responses, however, were reverted to growth arrest responses upon titration of cellular phospho-ERK1/2 levels by the MEK1/2 inhibitor AZD6244. These data suggest that a cellular threshold for active ERK1/2 levels exists and affects the cell fate between death and growth arrest. We also found that death-mediating ability of ERK2 is abolished by the catalytic site-disabling Lys52Arg replacement or significantly attenuated by the F-site recruitment site-disabling Tyr261Asn replacement, although unaffected by the mutations that disable the common docking groove or the dimerization interface. Therefore, ERK1/2 mediates death signaling dependently of kinase activity and specific physical interactions. Intriguingly, Tyr261Asn-replaced ERK2 could still mediate growth arrest signaling, further contrasting the molecular basis of ERK1/2-mediated growth arrest and death signaling. These data reveal a mechanism underlying the role of ERK1/2 as a focal point of Raf/MEK/ERK-mediated growth arrest and death signaling.

Four thiol peroxidases contain a conserved GCT catalytic motif and act as a versatile array of lipid peroxidases in Anabaena sp. PCC7120.

The Anabaena sp. (ANASP) genome contains seven open reading frames with homology to thiol peroxidase (TPx), also known as peroxiredoxin (Prx). Based on sequence similarities among putative TPx's derived from various cyanobacteria genomes, we designated the seven putative TPx members as VCP, VCT, TCS, and GCT clusters according to the sequence of their conserved catalytic motif. The GCT cluster consists of four members, named GCT1, GCT2, GCT3, and GCT4. The ANASP GCT-TPx genes were recombinantly expressed in Escherichia coli. The purified proteins were characterized with an emphasis on the ability to destroy various peroxides, the electron donor, and the conserved cysteine structure as a catalytic intermediate. All GCT members, as an atypical 2-Cys TPx family, exerted the highest peroxidase activity toward a lipid hydroperoxide using an electron from thioredoxin. Periplasmic protein analysis revealed that GCT2 and GCT4 are distributed in the cytoplasm, whereas GCT1 and GCT3, homologues of E. coli bacterioferritin comigratory protein/plant PrxQ, are localized in the periplasmic space. Immunoblots of the heterocystic proteins showed that the level of GCT2 in the heterocyst is comparable to that in the vegetative cell, whereas the other GCT members were not significantly detected in the heterocyst. The transcriptional responses of ANASP GCT genes to various oxidative stresses and growth environments were multifarious. Their intrinsic differences in transcriptional responsiveness and cellular localization suggest that this large GCT cluster is designed as an adaptive strategy to efficiently combat lipid hydroperoxide in Anabaena sp. that perform oxygenic photosynthesis and N(2) fixation.

Steady-State Levels of Phosphorylated Mitogen-Activated Protein Kinase Kinase 1/2 Determined by Mortalin/HSPA9 and Protein Phosphatase 1 Alpha in KRAS and BRAF Tumor Cells.

Although deregulation of MEK/extracellular signal-regulated kinase (ERK) activity is a key feature in cancer, high-magnitude MEK/ERK activity can paradoxically induce growth inhibition. Therefore, additional mechanisms may exist to modulate MEK/ERK activity in favor of tumor cell proliferation. We previously reported that mortalin/HSPA9 can facilitate proliferation of certain KRAS and BRAF tumor cells by modulating MEK/ERK activity. In this study, we demonstrated that mortalin can regulate MEK/ERK activity via protein phosphatase 1α (PP1α). We found that PP1α inhibition increases steady-state levels of phosphorylated MEK1/2 in various tumor cells expressing B-RafV600E or K-RasG12C/D Intriguingly, coimmunoprecipitation and in vitro binding assays revealed that mortalin facilitates PP1α-mediated MEK1/2 dephosphorylation by promoting PP1α-MEK1/2 interaction in an ATP-sensitive manner. The region spanning Val482 to Glu491 in the substrate-binding cavity and the substrate lid of mortalin were necessary for these physical interactions, which is consistent with conventional heat shock protein 70 (HSP70)-client interaction mechanisms. Nevertheless, mortalin depletion did not affect cellular PP1α levels or its regulatory phosphorylation, suggesting a nonconventional role for mortalin in promoting PP1α-MEK1/2 interaction. Of note, PP1α was upregulated in human melanoma and pancreatic cancer biopsy specimens in correlation with mortalin upregulation. PP1α may therefore have a role in tumorigenesis in concert with mortalin, which affects MEK/ERK activity in tumor cells.

Vandetanib and cabozantinib potentiate mitochondria-targeted agents to suppress medullary thyroid carcinoma cells.

Although the FDA-approved receptor tyrosine kinases inhibitors, vandetanib and cabozantinib, are used to treat surgically inoperable progressive medullary thyroid carcinoma (MTC), not all patients are responsive while the disease sometimes progresses after an initial response. To better understand MTC drug resistance at molecular and biochemical levels, we have generated drug-resistant subpopulations of the human MTC cell lines, TT and MZ-CRC-1, via prolonged exposure to vandetanib and cabozantinib. These drug-resistant progenies exhibited substantial cross-resistance to vandetanib and cabozantinib, suggesting that these inhibitors may invoke an overlapping resistance mechanism(s) in MTC cells. Of note, vandetanib and cabozantinib increased mitochondrial membrane potential (Δψm) in drug-naïve as well as drug-resistant cells but only drug-naïve cells exhibited substantially altered oxygen consumption and extracellular acidification rates. Therefore, these inhibitors appear to cause a bioenergetics stress to which drug-resistant MTC cells are more tolerant. Given the ability of vandetanib and cabozantinib to increase Δψm, we hypothesized that these inhibitors can augment growth inhibitory effects of mitochondria-targeted carboxy-proxyl and ubiquinone by increasing their Δψm-dependent uptake/retention in MTC cells. Indeed, our in vitro and mouse xenograft data strongly support this possibility.

Suppression of B-RafV600E melanoma cell survival by targeting mitochondria using triphenyl-phosphonium-conjugated nitroxide or ubiquinone.

Most BRAF-mutated melanomas initially responsive to the FDA-approved inhibitors preferentially targeting B-Raf mutated in Val600 residue eventually relapse, requiring additional therapeutic modalities. Recent studies report the significance of metabolic reprograming in mitochondria for maintenance of BRAF-mutated melanomas and for development of their drug resistance to B-Raf inhibitors, providing a rationale for targeting mitochondria as a potential therapeutic strategy for melanoma. We therefore determined whether mitochondria-targeted metabolism-interfering agents can effectively suppress human B-RafV600E melanoma cell lines and their dabrafenib/PLX4032-resistant progenies using mitochondria-targeted carboxy-proxyl (Mito-CP) and ubiquinone (Mito-Q). These agents exhibited comparable efficacy to PLX4032 in suppressing SK-MEL28, A375, and RPMI-7951 cells in vitro. As determined in SK-MEL28 and A375 cells, Mito-CP induced apoptotic cell death mediated by mitochondrial membrane depolarization and subsequent oxidative stress, which PLX4032 could not induce. Of note, Mito-CP also effectively suppressed PLX4032-resistant progenies of SK-MEL28 and A375. Moreover, when orally administered, Mito-CP suppressed SK-MEL28 xenografts in mice as effectively as PLX4032 without serious adverse effects. These data demonstrate that mitochondria-targeted agents have therapeutic potential to effectively suppress BRAF-mutated melanomas via an effect(s) distinct from those of B-Raf inhibitors.

Specific protein interaction of human Pag with Omi/HtrA2 and the activation of the protease activity of Omi/HtrA2.

The human PAG gene product (hPag), one member of the TSA/AhpC family, is overexpressed by oxidative stress, which causes apoptosis. To investigate the apoptotic signal transduction mediated by hPag, hPag-binding protein was screened using the yeast two-hybrid system. Omi/HtrA2 was identified as the hPag-binding protein. Omi/HtrA2, a potent proapoptotic factor, is released from the mitochondria into the cytoplasm as the mature form showing serine protease activity during apoptosis in response to oxidative stress. We found that hPag was able to interact with the mature form of Omi/HtrA2, not with the precursor form of Omi/HtrA2. The binding of Omi/HtrA2 to hPag was shown to involve the PDZ-binding domain in Omi/HtrA2. Also, the carboxyl-terminal domain of hPag was shown to be critical for the protein interaction. Using the yeast two-hybrid system and in vitro binding assay, the reduced form of hPag was able to interact with Omi/HtrA2. Interestingly, the protease activity given by the mature form of Omi/HtrA2 was significantly activated by the binding to hPag. Taken together, these results suggest that the specific protein interaction may participate as a molecular switch in modulating cell death in response to oxidative stress.