Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival.

BACKGROUND: Pancreatic cancer is a leading cause of cancer-related deaths. Increased activity of the epidermal growth factor receptor (EGFR) is often observed in pancreatic cancer, and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration. Nevertheless, erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes. We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential (Δψm), facilitate tumor cell uptake of Δψm-sensitive agents, disrupt mitochondrial homeostasis, and subsequently trigger tumor cell death. Erlotinib has not been tested for this effect. AIM: To determine whether erlotinib can elevate Δψm and increase tumor cell uptake of Δψm-sensitive agents, subsequently triggering tumor cell death. METHODS: Δψm-sensitive fluorescent dye was used to determine how erlotinib affects Δψm in pancreatic adenocarcinoma (PDAC) cell lines. The viability of conventional and patient-derived primary PDAC cell lines in 2D- and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone (MitoQ), a Δψm-sensitive MitoQ. The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0. The preclinical efficacy of the two-drug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts. RESULTS: Erlotinib elevated Δψm in PDAC cells, facilitating tumor cell uptake and mitochondrial enrichment of Δψm-sensitive agents. MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses, while erlotinib pretreatment potentiated low doses of MitoQ. SynergyFinder suggested that these drugs synergistically induced tumor cell lethality. Consistent with in vitro data, erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent. CONCLUSION: Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Selpercatinib combination with the mitochondria-targeted antioxidant MitoQ effectively suppresses RET-mutant thyroid cancer.

Genetic alternation of REarranged during Transfection (RET) that leads to constitutive RET activation is a crucial etiological factor for thyroid cancer. RET is known to regulate mitochondrial processes, although the underlying molecular mechanisms remain unclear. We previously showed that the multi-kinase inhibitors vandetanib and cabozantinib increase the mitochondrial membrane potential (Δψm) in RET-mutated thyroid tumor cells and that this effect can be exploited to increase mitochondrial enrichment of Δψm-sensitive agents in the tumor cells. In this study, we hypothesized that the RET-selective inhibitor, selpercatinib, can increase Δψm and, subsequently, tumor cell uptake of the mitochondria-targeted ubiquinone (MitoQ) to the level to break the mitochondrial homeostasis and induce lethal responses in RET-mutated thyroid tumor cells. We show that selpercatinib significantly increased Δψm, and its combination with MitoQ synergistically suppressed RET-mutated human thyroid tumor cells, which we validated using RET-targeted genetic approaches. Selpercatinib and MitoQ, in combination, also suppressed CCDC6-RET fusion cell line xenografts in mice and prolonged animal survival more effectively than single treatments of each agent. Moreover, we treated two patients with CCDC6-RET or RETM918T thyroid cancer, who could not take selpercatinib at regular doses due to adverse effects, with a dose-reduced selpercatinib and MitoQ combination. In response to this combination therapy, both patients showed tumor reduction. The quality of life of one patient significantly improved over a year until the tumor relapsed. This combination of selpercatinib with MitoQ may have therapeutic potential for patients with RET-mutated tumors and intolerant to regular selpercatinib doses.

eIF5A-Independent Role of DHPS in p21CIP1 and Cell Fate Regulation.

Deoxyhypusine synthase (DHPS) catalyzes the first step of hypusination of the elongation translation factor 5A (eIF5A), and these two proteins have an exclusive enzyme-substrate relationship. Here we demonstrate that DHPS has a role independent of eIF5A hypusination in A375 and SK-MEL-28 human melanoma cells, in which the extracellular signal regulated kinase 1/2 (ERK1/2) pathway is deregulated. We found that RNA interference of DHPS induces G0/G1 cell cycle arrest in association with increased p21CIP1 expression in these cells whereas eIF5A knockdown induces cell death without increasing p21CIP1 expression. Interestingly, p21CIP1 knockdown switched DHPS knockdown-induced growth arrest to cell death in these cells, suggesting a specific relation between DHPS and p21CIP1 in determining cell fate. Surprisingly, ectopic expression of DHPS-K329R mutant that cannot hypusinate eIF5A abrogated DHPS knockdown-induced p21CIP1 expression in these cells, suggesting a non-canonical role of DHPS underlying the contrasting effects of DHPS and eIF5A knockdowns. We also show that DHPS knockdown induces p21CIP1 expression in these cells by increasing CDKN1A transcription through TP53 and SP1 in an ERK1/2-dependent manner. These data suggest that DHPS has a role independent of its ability to hypusinate eIF5A in cells, which appears to be important for regulating p21CIP1 expression and cell fate.

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.

Growth Inhibitory Signaling of the Raf/MEK/ERK Pathway.

In response to extracellular stimuli, the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway regulates diverse cellular processes. While mainly known as a mitogenic signaling pathway, the Raf/MEK/ERK pathway can mediate not only cell proliferation and survival but also cell cycle arrest and death in different cell types. Growing evidence suggests that the cell fate toward these paradoxical physiological outputs may be determined not only at downstream effector levels but also at the pathway level, which involves the magnitude of pathway activity, spatial-temporal regulation, and non-canonical functions of the molecular switches in this pathway. This review discusses recent updates on the molecular mechanisms underlying the pathway-mediated growth inhibitory signaling, with a major focus on the regulation mediated at the pathway level.

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.

Treatment of Cells and Tissues with Chromate Maximizes Mitochondrial 2Fe2S EPR Signals.

In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria.

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.

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.

MEK1/2 Inhibitors: Molecular Activity and Resistance Mechanisms.

Aberrant activation of the three-layered protein kinase cascade, Raf/MEK/ERK, is often detected in human cancer, which is mainly attributed to the oncogenic alterations of RAF, or its upstream activators RAS or cell surface receptor tyrosine kinases. Deregulated activity of the Raf/MEK/ERK pathway drives uncontrolled tumor cell proliferation and survival, thus providing a rational therapeutic target for the treatment of many cancers. While Raf, MEK1/2, and ERK1/2 are equally important targets for the design of therapeutic small molecular weight inhibitors, the effort to develop MEK1/2-specific inhibitors has been greatly successful. Particularly, MEK1/2 have been relatively advantageous for the design of highly selective adenosine triphosphate (ATP)-noncompetitive inhibitors. Indeed, a plethora of highly selective and potent MEK1/2 inhibitors are now available and many of those inhibitors have been evaluated for their therapeutic potential. Herein, we review different MEK1/2 inhibitors that have been studied for their inhibitory mechanisms and therapeutic potential in cancer. Some of the key structural features of MEK1/2 that are important for the efficacy of these inhibitors are also discussed. In addition, we discuss current challenges and future prospective in using these advanced MEK1/2 inhibitors for cancer therapy.

ERK1/2 can feedback-regulate cellular MEK1/2 levels.

Signal transduction of the Raf/MEK/ERK pathway is regulated by various feedback mechanisms. Given the greater molar ratio between Raf-MEK than between MEK-ERK in cells, it may be possible that MEK1/2 levels are regulated to modulate Raf/MEK/ERK activity upon pathway stimulation. Nevertheless, it has not been reported whether MEK1/2 expression can be subject to a feedback regulation. Here, we report that the Raf/MEK/ERK pathway can feedback-regulate cellular MEK1 and MEK2 levels. In different cell types, ΔRaf-1:ER- or B-Raf(V600E)-mediated MEK/ERK activation increased MEK1 but decreased MEK2 levels. These regulations were abrogated by ERK1/2 knockdown mediated by RNA interference, suggesting the presence of a feedback mechanism that regulates MEK1/2 levels. Subsequently, analyses using qPCR and luciferase reporters of the DNA promoter and 3' untranslated region revealed that the feedback MEK1 upregulation was in part attributed to increased transcription. However, the feedback MEK2 downregulation was only observed at protein levels, which was blocked by the proteasome inhibitors, MG132 and bortezomib, suggesting that the MEK2 regulation is mediated at a post-translational level. These results suggest that the Raf/MEK/ERK pathway can feedback-regulate cellular levels of MEK1 and MEK2, wherein MEK1 levels are upregulated at transcriptional level whereas MEK2 levels are downregulated at posttranslational level.

Active ERK2 is sufficient to mediate growth arrest and differentiation signaling.

Although extracellular signal-regulated kinases (ERK1/2) have been shown to be required in Raf/MEK/ERK pathway signaling, its sufficiency for mediating the pathway signaling has not been firmly established. In an effort to address this, we evaluated previously described ERK2 mutants that exhibit enhanced autophosphorylation of TEY sites in the activation loop in terms of their ability to induce growth arrest and differentiation in LNCaP and PC12 cells. We demonstrate that expression of ERK2-L73P/S151D, containing Lys73Pro and Ser151Asp substitutions that synergistically promote ERK autophosphorylation, is sufficient to induce growth arrest and differentiation, whereas expression of ERK2-I84A and ERK2-R65S/D319N is not as effective. When compared to the constitutively active MEK1-ΔN3/S218E/S222D, expression of ERK2-L73P/S151D only mildly increased ERK kinase activity in cells, as assessed using the ERK substrates p90(RSK) and ETS domain-containing protein (ELK1). However, ERK2-L73P/S151D expression effectively induced down-regulation of androgen receptors, Retinoblastoma (Rb) protein and E2F1 transcription factor, and up-regulation of p16(INK4A) and p21(CIP1), accompanied by cell-cycle arrest and morphological differentiation in LNCaP cells and neurite-like processes in PC12 cells. These effects and the TEY site phosphorylation of ERK2-L73P/S151D were abrogated upon introduction of the active site-disabling Lys52Arg mutation, suggesting that its autoactivation drives this signaling. Moreover, introduction of mutations Asp316/319Ala or Asp319Asn, which impair the common docking site/D-domain-based physical interaction of ERK, did not significantly affect ERK2-L73P/S151D signaling, suggesting that ERK2 mediates growth arrest and differentiation independently of the conventional ERK-target interaction mechanism. Thus, our study presents convincing evidence of ERK sufficiency for Raf/MEK/ERK signaling.

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.

A mortalin/HSPA9-mediated switch in tumor-suppressive signaling of Raf/MEK/extracellular signal-regulated kinase.

Dysregulated Raf/MEK/extracellular signal-regulated kinase (ERK) signaling, a common hallmark of tumorigenesis, can trigger innate tumor-suppressive mechanisms, which must be inactivated for carcinogenesis to occur. This innate tumor-suppressive signaling may provide a potential therapeutic target. Here we report that mortalin (HSPA9/GRP75/PBP74) is a novel negative regulator of Raf/MEK/ERK and may provide a target for the reactivation of tumor-suppressive signaling of the pathway in cancer. We found that mortalin is present in the MEK1/MEK2 proteome and is upregulated in human melanoma biopsy specimens. In different MEK/ERK-activated cancer cell lines, mortalin depletion induced cell death and growth arrest, which was accompanied by increased p21(CIP1) transcription and MEK/ERK activity. Remarkably, MEK/ERK activity was necessary for mortalin depletion to induce p21(CIP1) expression in B-Raf(V600E)-transformed cancer cells regardless of their p53 status. In contrast, in cell types exhibiting normal MEK/ERK status, mortalin overexpression suppressed B-Raf(V600E)- or ΔRaf-1:ER-induced MEK/ERK activation, p21(CIP1) expression, and cell cycle arrest. Other HSP70 family chaperones could not effectively replace mortalin for p21(CIP1) regulation, suggesting a unique role for mortalin. These findings reveal a novel mechanism underlying p21(CIP1) regulation in MEK/ERK-activated cancer and identify mortalin as a molecular switch that mediates the tumor-suppressive versus oncogenic result of dysregulated Raf/MEK/ERK signaling. Our study also demonstrates that p21(CIP1) has dual effects under mortalin-depleted conditions, i.e., mediating cell cycle arrest while limiting cell death.

The Predicted Proteomic Network Associated with the Antiarthritic Action of Qingfu Guanjieshu in Collagen-II-Induced Arthritis in Rats.

Qingfu Guanjieshu (QFGJS) is an herbal preparation for treating rheumatoid arthritis (RA). Previous studies revealed that QFGJS significantly inhibited experimental arthritis and acute inflammation, accompanied by reduction of proinflammatory cytokines and elevation of anti-inflammatory cytokines. This study aims to identify the targeted proteins and predict the proteomic network associated with the drug action of QFGJS by using 2D gel and MALDI-TOF-MS/MS techniques. Thirty female Wistar rats were evenly grouped as normal and vehicle- and QFGJS-treated CIA rats. The antiarthritic effect of QFGJS was examined with a 19-day treatment course, and the knee synovial tissues of animals from each group were obtained for 2D gel and MALDI-TOF-MS/MS analysis. Results showed that QFGJS significantly ameliorated collagen II-induced arthritis when administrated at 2.8 g/kg body weight for 19 days. 2D gel image analysis revealed 89 differentially expressed proteins in the synovial tissues among the normal and vehicle- and QFGJS-treated CIA rats from over 1000 proteins of which 63 proteins were identified by MALDI-TOF-MS/MS analysis, and 32 proteins were included for classification of functions using Gene Ontology (GO) method. Finally, 14 proteins were analyzed using bioinformatics, and a predicted proteomic network related to the anti-arthritic effect of QFGJS was established, and Pgk1 plays a central role.

Chemical and DNA authentication of taste variants of Gynostemma pentaphyllum herbal tea.

Gynostemma pentaphyllum Makino (Gp) was once used as a sweetener in Japan and is now widely consumed as an herbal tea worldwide for lowering cholesterol levels. Two taste variants, bitter and sweet, of Gp exist in the commercial market, but they cannot be differentiated morphologically nor by existing chemical analytical methods. This has been creating a problem in quality control of Gp products. In the present study, using HPLC-DAD and HPLC-ESI-MS analysis, we found that the Gp saponins, not flavonoids, from the sweet and bitter variants have distinctly different profiles. In addition, the two variants share only 69.01% homology in the ribosomal ITS-1 region, suggesting a phylogenic gap between these two variants. The combinations of chemical profiling and phylogenic analysis clearly confirm, for the first time, the distinction between these two taste variants. This information has direct application in the authentication and quality assessment of the various Gynostemma tea products.

Oleanolic acid isolated from Oldenlandia diffusa exhibits a unique growth inhibitory effect against ras-transformed fibroblasts.

AIMS: Oldenlandia diffusa (Willd.) Roxb. (O. diffusa) is a commonly used traditional Chinese medicine for treating cancer. Its pharmacological activities and anti-cancer effects have been the focus of intense research in recent years. In the present study, we aim to investigate whether the five major compounds from O. diffusa possess a unique inhibitory activity against ras-transformed cells in a well-established cell model. MAIN METHODS: The anti-cancer effects of O. diffusa were assessed in a co-culture system containing normal and transformed Rat 6 (R6) fibroblasts. In addition, a transwell assay was used to examine the interaction between the drugs and the co-cultivated cells. KEY FINDINGS: Our data showed that among the samples tested, oleanolic acid (OA), but not the structural isomer ursolic acid (UA), inhibits the growth of ras oncogene-transformed R6 cells at a dosage that is not toxic to the co-cultivated normal fibroblasts. A significant inhibitory effect was also observed in the transwell experiments, indicating that the mode of action for OA-mediated growth inhibition of transformed cells does not require direct cell-to-cell contact between normal and ras-transformed cells. Data obtained from experiments conducted with the conditioned medium that was collected from normal R6 cells treated with OA also suggest that OA might cause normal cells to secrete inhibitory factor(s) against the transformed cells. The enhanced ability of OA to cause cytotoxicity in transformed cells in the presence of normal fibroblasts is also observed with the human hepatocellular carcinoma cell line, SMMC-7721. SIGNIFICANCE: The present study demonstrates that OA may possess both cancer chemotherapeutic and chemopreventive activities. Thus, it may have great potential for clinical application as a novel anti-cancer drug.