Increase In Mitochondrial Membrane Permeability Research Articles

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

Mannose delays senescence in broccoli florets via maintaining mitochondrial morphological structure and function

The influence of mannose treatment on mitochondrial physiological parameters was investigated to postpone broccoli senescence. The results showed that mannose treatment could reduce the level of mitochondrial reactive oxygen species (ROS) to slow down the damage to its ultrastructure. Mannose inhibited malondialdehyde (MDA) accumulation and the decline of cytochrome c/a (Cyt c/a), and delayed the increase of mitochondrial membrane permeability. It also alleviated the over-opening of the mitochondrial membrane permeability transition pore (MPTP) by regulating expressions of MPTP component genes such as BoVDAC, BoHXK and BoANT. Moreover, mannose treatment improved the activities of mitochondrial antioxidant enzymes, including ascorbate oxidase (APX), superoxide dismutase (SOD), catalase (CAT), and ascorbic acid (AsA) content. Also, it increased the activities of succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and Ca2+-ATPase, which were crucial for the mitochondrial energy metabolism. Taken together, our data indicated that mannose treatment inhibited the senescence of broccoli by maintaining the mitochondrial morphological structure and function.

Mechanisms of Mitochondrial Apoptosis-Mediated Meat Tenderization Based on Quantitative Phosphoproteomic Analysis.

This study investigates the mechanism of phosphorylation in the regulation of apoptosis-mediated meat tenderization during postmortem aging. The results found that the pork muscle exhibited apoptotic potential at early postmortem (48 h) and showed more tenderness at late postmortem, as evidenced by the increase in mitochondrial membrane permeability (MMP), Ca2+ level, reactive oxygen species (ROS) content, and caspases activity at 0 h to 48 h, and decreases in ATP level at 0 h to 24 h and shear force at 12 h to 120 h (p < 0.05). Phosphoproteomic analysis revealed that phosphorylation regulated apoptosis by modulating ATP and calcium bindings as well as apoptotic signaling, which occurred within early 12 h and mainly occurred at 12 h to 48 h postmortem. Moreover, differential expression of phosphoproteins demonstrated that phosphorylation regulated oxidative stress-induced apoptosis and rigor mortis, thereby promoting the development of meat tenderness. Our results provide insights into the roles of phosphorylation in various physiological processes that affect meat tenderness.

Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α-Ve-Cadherin Pathway.

The objective of this study was to investigate the molecular response to damage at the blood-brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases.

Suppression of DRP1‑mediated mitophagy increases the apoptosis of hepatocellular carcinoma cells in the setting of chemotherapy.

The efficacy of chemotherapy for hepatocellular carcinoma (HCC) remains unsatisfactory, primarily due to inherent self‑defense mechanisms (e.g., mitophagy and autophagy). In the present study, we aimed to explore the pro‑apoptotic effects of targeting mitophagy to potentiate the efficacy of chemotherapy for HCC. HCC cells were subjected to cisplatin, after which cisplatin‑induced mitophagy was quantified by immunofluorescence. Mdivi‑1, a specific dynamin‑related protein1 (DRP1) inhibitor, was used to study the role of DRP1 in cisplatin‑induced HCC mitophagy. The synergistic effect of cisplatin and the DRP1 inhibitor on HCC was assessed invitro and invivo. Accordingly, cisplatin induced mitophagy in surviving HCC cells by activating DRP1. The DRP1 inhibitor (Mdivi‑1) increased the apoptosis of cisplatin‑treated HCC cells by targeting mitophagy. Mechanistically, Mdivi‑1 upregulated Bax and downregulated Bcl‑xL, leading to an increase in mitochondrial membrane permeability and subsequent release of cytochromec from mitochondria into the cytosol, thereby aggravating cisplatin‑induced apoptosis in HCC cells. Moreover, Mdivi‑1 acted synergistically with cisplatin to suppress HCC xenograft growth invivo. Our results indicate that targeting cisplatin‑mediated mitophagy increases HCC apoptosis via DRP1 inhibition, providing preclinical proof of concept for combination therapy targeting mitophagy to potentiate the efficacy of chemotherapy.

Ginkgolide K attenuates neuronal injury after ischemic stroke by inhibiting mitochondrial fission and GSK-3β-dependent increases in mitochondrial membrane permeability.

Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3β was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3β translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3β phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3β. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3β-induced mPTP opening. Treating mice with GK prevented GSK-3β and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

Identification of a novel compound that inhibits both mitochondria-mediated necrosis and apoptosis

In various pathological events, particularly in oxygen radical-mediated cell injury, both apoptosis and necrosis play essential roles. Apoptosis and some types of necrosis are induced via increases in mitochondrial membrane permeability, called mitochondrial outer membrane permeabilization (MOMP) and permeability transition pore (PTP) opening, respectively. To search for small compounds that inhibit both MOMP-mediated apoptosis and PTP-mediated necrosis, we performed a mitochondria-based high-throughput screening of a chemical library. We identified TMD#7538, a small compound that inhibits both MOMP and PTP opening. Consistent with the fact that this compound inhibited both apoptosis and necrosis, it efficiently suppressed H2O2-induced cell death in mouse embryonic fibroblasts and rat neonatal cardiomyocytes.

Abstract POSTER-THER-1422: Development of proteomic biomarkers for the apoptosis pathway in ovarian cancer cell lines and determination of the appropriate sequence of the SMAC-mimetic birinapant (TL32711) and docetaxel for optimal therapeutic effect

Abstract Background: The SMAC-mimetic birinapant (TL32711) induces apoptosis of ovarian cancer (OC) cell lines by eliminating the inhibitors of apoptosis (IAPs) and attenuating NFkB signaling. The increase in mitochondrial membrane permeability (MOMP) induced by docetaxel releases cytochrome c, unleashing the caspase cascade, which culminates in apoptosis. In addition, caspase-8 cleavage results in activation of the BH3-only protein BH3-interacting domain death agonist (BID), the product of which (truncated BID; tBID) is required for death receptor-induced apoptosis via BID activation and MOMP. Thus, caspase-8 is at the intersection between the extrinsic and intrinsic apoptosis pathways. We hypothesized that the combination of birinapant and docetaxel would be synergistic augmenting the apoptotic response in OC. Our aim was to determine the most appropriate sequence for optimal therapeutic effect in a cell line model and develop potential proteomic biomarkers. Methods: Toxicity of the SMAC-mimetic birinapant and docetaxel was assessed in a panel of OC cell lines using XTT assay with specific attention to the order and timing of drug administration. The target protein changes were assessed using Western blot and Simple Western. Similar protein changes are being assessed in mouse model. Cytokine analysis was performed on the secretome of cells treated with drug. Results: There was variable sensitivity to single-agent exposure to the small molecule SMAC mimetic in OC cell lines. Increased cell death was seen upon administration of both docetaxel and birinapant, with enhanced toxicity when birinapant was administered prior to docetaxel. Degradation of cIAP1, the main pharmacodynamic outcome of brianapant, was seen after one hour of birinapant exposure, and was maintained up to 12 hours. Interestingly, cIAP2 levels increased from 3 hours to 24 hours. Cleavage of caspase-3 began at 3 hours and was maximal at 24 hours. PARP cleavage occurred 24 hours after treatment. Docetaxel induced cleavage of BID. Enhanced caspase-3 and caspase-8 cleavage were seen when both birinapant and doctaxel were administered. Similar protein changes are being examined in ongoing mouse experiment and will be reported at the meeting. Conclusions: The combination of birinapant and docetaxel results in enhanced cell death in an OC cell line model with evidence of target protein engagement. Citation Format: Anne M. Noonan, Lidia Hernandez, Michelle Herrmann, Jinqui Chen, Christina M. Annunziata. Development of proteomic biomarkers for the apoptosis pathway in ovarian cancer cell lines and determination of the appropriate sequence of the SMAC-mimetic birinapant (TL32711) and docetaxel for optimal therapeutic effect [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1422.

Lipid Rafts, Endoplasmic Reticulum and Mitochondria in the Antitumor Action of the Alkylphospholipid Analog Edelfosine

The so-called alkylphospholipid analogs (APLs) constitute a family of synthetic antitumor compounds that target cell membranes. The ether phospholipid edelfosine has been considered the long-standing prototype of these antitumor agents and promotes apoptosis in tumor cells by a rather selective way, while sparing normal cells. Increasing evidence suggests that edelfosine-induced apoptosis involves a number of subcellular structures in tumor cells, including plasma membrane lipid rafts, endoplasmic reticulum (ER) and mitochondria. Edelfosine has been shown to accumulate in plasma membrane lipid rafts, ER and mitochondria in different tumor cells in a cell type-dependent way. Edelfosine induces apoptosis in several hematopoietic cancer cells by recruiting death receptor and downstream apoptotic signaling molecules into lipid rafts and displacing survival signaling molecules from these membrane domains. However, in vitro and in vivo evidences suggest that edelfosine-induced apoptosis in solid tumor cells is mediated through an ER stress response. Both raft- and ER-mediated proapoptotic responses require a mitochondrial-related step to eventually promote cell death, and overexpression of Bcl-2 or Bcl-xL prevents edelfosine-induced apoptosis. Edelfosine can also interact with mitochondria leading to an increase in mitochondrial membrane permeability and loss of mitochondrial membrane potential. Edelfosine treatment also induced a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. The involvement of lipid rafts, ER and mitochondria in the apoptotic response induced by edelfosine may provide new avenues for targeting cancer cells as well as new opportunities for cancer therapy.

Conditioned Media of Human Umbilical Cord Blood Mesenchymal Stem Cell-derived Secretome Induced Apoptosis and Inhibited Growth of HeLa Cells

BACKGROUND: Secreted factors contained in conditioned media (CM) of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) known as secretome, was suspected to have important roles in regulating cells. This study was conducted to investigate the role of CM-hUCB-MSCs-derived secretome in apoptosis and growth of HeLa cells.METHODS: HeLa cells were treated with secretome in various concentrations (0, 0.2, 2 and 20%) for 24 and 48 hours. Trypan blue exclusion assay was performed to detect cell viability. Meanwhile sub-G1 apoptotic assay was performed to detect apoptotic cells. The transition of mitochondrial transmembrane potential (TMP), which occurs in the apoptotic process, was analyzed by mitochondrial membrane potential (ΔΨM) assay. Both sub-G1 and ΔΨM assays were performed using FACSCanto flow cytometer. Statistical analyses were conducted using IBM SPSS Statistics to detect significance level at p&lt;0.05.RESULTS: Secretome significantly induced cell death starting at concentration of 0.2% within a 24-hour period (p&lt;0.05). Secretome significantly induced cell death in concentration and time dependent manner (p&lt;0.05). The cell death was then confirmed as apoptosis through sub-G1 analysis. Due to the underlying apoptotic mechanism, we found distinct decrease of TMP, indicating an increase in mitochondrial membrane permeability of HeLa cells. In addition, we found that HeLa cell growth was inhibited partially by secretome.CONCLUSION: Taken together, we conclude that CMhUCB-MSCs-derived secretome significantly induced apoptosis of HeLa cells in a concentration and time dependent manner through mitochondrial apoptotic pathway. The secretome might also play important role in inhibiting HeLa cell growth.KEYWORDS: umbilical cord blood, mesenchymal stem cell, secretome, apoptosis, growth, cancer

The investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL cellsin vitro, as a single agent and in combination with other drugs

Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in the U.S. The course of the disease has been shown to be negatively impacted by increased levels of BCL2. Strategies to downregulate BCL2 and shift the balance towards cellular demise are actively being explored. Therefore, we examined whether the investigational agent MLN2238 could inhibit the proteasomal machinery and induce CLL cell death while also downregulating BCL2. MLN2238-induced cell death was studied in peripheral blood mononuclear cells from 28 CLL patients. MLN2238 produced a dose-dependent reduction in BCL2 and CLL cell viability with maximum cell death observed at a 50nmol/l concentration by 48h. Annexin-V staining, PARP1 and caspase-3 cleavage along with an increase in mitochondrial membrane permeability were noted after cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. Furthermore, we observed enhanced anti-CLL effects in tumour cells treated with either a combination of MLN2238 and the BH3 mimetic AT-101 or MLN2238 and fludarabine. Together, our data suggest the potential for proteasome inhibitor based therapy in CLL and the rationale design of drug combination strategies based on CLL biology.

The Novel Proteasome Inhibitor MLN2238 (Ixazomib) Induces Death In CLL Cells In Vitro and Potentiates Lethality Of Fludarabine and The BCL2 Inhibitor At-101

BackgroundChronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells in the peripheral blood, bone marrow, lymph nodes, and spleen. This is due to a combined effect of deferred apoptosis with slow, but persistent proliferation of malignant cells. In CLL, tumor-sustaining homeostasis is critically maintained by the ubiquitin proteasome system. The proteasome mediates degradation of various transcription factors such as TP53 as well as upholding a balance between the anti and pro apoptotic proteins of the BCL2 family. Previous studies have demonstrated that the clinical course of the disease is negatively associated with malfunctioning apoptotic pathways that result in increased levels of BCL2. Thus, identification and correction of defects that affect programmed cell death offer therapeutic vantage to reset and engage cell death pathways in CLL. AimExamination of the anti-CLL properties of the investigational agent MLN2238 (Millennium Pharmaceuticals, Inc., Cambridge, MA) and its ability to inhibit the proteasomal machinery; induce CLL cell death and downregulate BCL2. MLN2238 activity was also investigated in conjunction with anti-CLL therapies such as fludarabine and dexamethasone along with the BH3 mimetic BCL2 inhibitor, AT-101 (Ascenta Pharmaceuticals, Malvern, PA). MethodsCLL cells with >90% CD19+ tumor population were obtained from 28 patients with a confirmed diagnosis of CLL. Proteasomal activity was measured using synthetic fluorogenic peptide substrates. Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. ResultsMLN2238 inhibited the chymotrypsin-like proteasomal activity by more than 90% (p<0.005) in all patient samples without altering PSMB5 protein levels. Moderate to minimal inhibitory effect on caspase-like and trypsin-like proteasomal activities, respectively, was also noted. CLL cells showed a concentration dependent decrease in viability in response to treatment with MLN2238 at an IC50 of 50 nM. MLN2238 treated cells underwent apoptosis in a dose dependent manner with a median dose effect (cell death) observed in 42% of cells at 25 nM (range 10% - 54%) and 60% of cells at a 50 nM concentration (range, 25% - 73%). PARP-1 and caspase-3 cleavage along with an increase in MOMP was also noted after CLL cells were treated with MLN2238; however, apoptosis was only partially blocked by the pan-caspase inhibitor z-VAD.fmk. BCL2 downregulation was dose-dependent and was observed as early as 12 hours. We sought to determine whether directly disrupting BCL2 function with AT-101 could enhance the anti-CLL effects of MLN2238. When used at sub-IC50 concentrations, AT1-10 synergized with MLN2238 to induce CLL cell death. Synergy was also observed when MLN2238 was paired with the cytotoxic agent fludarabine, whereas the combination of MLN2238 and dexamethasone resulted in additive anti-CLL activity. ConclusionWhile PI have made an important impact in various B cell cancers, their role in CLL has not been well established. We investigated preclinically, a novel PI and noted that targeting the proteasome with MLN2238 resulted in lethal events in CLL cells, which were further enhanced by disruption of the BCL2 prosurvival pathway. Moreover, proteasome disruption sensitized CLL cells to the cytotoxic effect of fludarabine, an important therapeutic in CLL. These data provides the mechanistic basis for evaluation of MLN2238 in CLL through rationale design of drug combination strategies based on CLL biology.We would like to acknowledge the Leukemia and Lymphoma Society (A.C.-K. is a Leukemia and Lymphoma Scholar in Clinical Research) for their ongoing support. We are also grateful to Mary Ella Mahoney Davidson (Millennium Pharmaceuticals) for providing logistical support. Disclosures:Foran:Celgene: Research Funding.

Interference Of The Tumor Supportive Effects Of BCL2 and MCL1 Sensitize Malignant Plasma Cells To The Lethal Effects Of Lenalidomide and Dexamethasone Regimen: An Important Clinical Path For BCL2 Targeting Drugs

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy in the U.S. and is characterized by increased immunoglobulin production and infiltration of the bone marrow by malignant plasma cells. Myeloma cell growth is supported by both the elements present in the bone marrow microenvironment as well as deregulation of internal cellular systems associated with proliferation and apoptosis. Defective programmed cell death by BCL2 or MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Various strategies to mitigate the effects of increased BCL2 functionality, via small molecule inhibitors or BCL2-specific antisense oligonucleotides have been previously examined in MM. These studies revealed that 1) apart from BCL2, additional anti-apoptotic members belonging to the BCL2 family (i.e. MCL1, BCL-xL) play integral roles in maintenance of the myeloma cell phenotype, 2) drugs, which target only BCL2 offer limited therapeutic advantage and 3) given that survival factors engaged in MM are multifactorial, perhaps isolated targeting of the BCL2 pathways may not be sufficient. We therefore hypothesized that the clinical development of drugs that target various antiapoptotic BCL2 family members will require a partnership with established anti-MM regimens whose activity can potentially be further enhanced with correction of the apoptotic response system. AimSince lenalidomide and dexamethasone (LD) is an established therapy for plasma cell cancers, we investigated if the pan-BCL2 inhibitor AT-101 (BH3 mimetic), which has high binding specificity for BCL2, MCL1 and BCL-xL, can be an effective therapeutic partner to enhance anti-MM effects of LD. MethodsHuman MM (KMS11, U266, OPM2) and WM (BCWM.1) cell lines along with their corresponding bortezomib resistant (BR) clones, (KMS11/BR, U266/BR, OPM2/BR and BCWM.1/BR) which were developed in our laboratory were used in this study. For gene expression profiling, the Illumina HumanHT-12 v3 whole-genome gene expression array and Nanostring nCounter mRNA quantification assays (NanoString, Seattle, WA) were utilized. Statistical analysis was conducted using R-based packages and the MeV software (TIGR). Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. ResultsGene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of various BCL2 family members, notably MCL1 and BCL-xL. In a dose-dependent manner, AT-101 was able to downregulate BCL2 and MCL1 and induced apoptotic cell death in MM and WM cells in a dose dependent manner. Tumor cell death was associated with caspase and PARP-1 cleavage accompanied by an increase in MOMP. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). Nanostring nCounter mRNA quantification and IPA analysis revealed differential changes in the CCNA, FRZB, FYN, IRF1, PTPN11 genes in LDA treated cells. IPA canonical pathway analysis demonstrated the p53 signaling and the cyclins and cell cycle regulation pathways to be the most significantly activated by LDA therapy. SummaryIn summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. This study lays the rationale for engaging and handicapping the intrinsic apoptotic system through the pan-BCL2 targeting capabilities of AT-101; thus enhancing the anti-MM effects of lenalidomide/dexamethasone. Disclosures:Foran:Celgene: Research Funding.

Anticancer (hexacarbonyldicobalt)propargyl aryl ethers: Synthesis, antiproliferative activity, apoptosis induction, and effect on cellular oxidative stress

While an increasing number of (hexacarbonyldicobalt)alkynes have been found to possess antiproliferative activity against a number of cancer cell lines, the role of the organometallic moiety in this bioactivity is not well understood. To gain a better understanding of cobalt's role in the medicinal chemistry of these compounds, several simplified analogs of a known organocobalt anticancer compound were synthesized and assessed for antiproliferative activity against MDA-MB-231 human breast cancer cells. These compounds, mostly (hexacarbonyldicobalt)propargyl aryl ethers, caused 45–93% growth inhibition of that cell line at 40μM in a 72h crystal violet staining assay. The most active analog was the organocobalt nitroaromatic ether 3a, with an IC50 of 3.3±0.9μM. Flow cytometric assays on the same cell line demonstrated that 3a strongly induces apoptosis, arrests the cell cycle at the S phase, increases cellular oxidative stress levels, and induces permeability of the mitochondrial membrane. While the non-cobalt-containing precursor to 3a also caused an increase in mitochondrial membrane permeability, it did not produce an increase in oxidative stress levels, nor did it have apoptosis-inducing or antiproliferative effects. The induction of oxidative stress in the cell may be responsible for some of the antiproliferative activity of compound 3a against this cell line.

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

Estrogen receptor negative (ER−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I2) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I2 (3μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER−ve mammary tumors could be sensitized to I2-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I2 treated MDA-MB231 cells. Further, CQ (20μM) in combination with I2, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I2 treatment alone. Flowcytometry of I2 and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p<0.01) and translocation of cathepsin D activity to cytosol relative to I2 treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I2 and CQ co-treated mice relative to I2 or vehicle treated mice. These data indicate that inhibition of autophagy renders ER−ve breast tumor cells more sensitive to I2 induced apoptosis. Thus, I2 together with autophagy inhibitor could have a potential tumorostatic role in ER−ve aggressive breast tumors that may be evaluated in future studies.

Cell apoptosis induced by zinc deficiency in osteoblastic MC3T3-E1 cells via a mitochondrial-mediated pathway

Deficiency of zinc plays an important role in the pathogenesis of osteoporosis; however, the underlying mechanism is not well understood. Apoptosis of osteoblast causing the loss of bone mass is an important event in the osteoporosis. In this article, we investigated whether zinc deficiency would induce cell apoptosis in MC3T3-E1 cells and ask if it is involved in mitochondrial-mediated pathway. Significant increased apoptosis were observed in zinc deficiency group (ZnD: 5 μM TPEN and 1 μM zinc) compared with untreated control or zinc adequacy group (ZnA: 5 μM TPEN and 15 μM zinc). The mitochondrial membrane potential was strikingly reduced in ZnD group. Furthermore, we observed that the levels of Bax in mitochondria fraction and cyto c, AIF, and cleaved caspase-3/-9 in cytosol fraction were increased in ZnD group. We proposed that zinc deficiency would induce the translocation of Bax into mitochondria, which could lead to the reduction in mitochondrial membrane potential as well as the increase in mitochondrial membrane permeability. In addition, cyto c and AIF were released from mitochondria into the cytosol, which finally activated caspase-dependent and caspase-independent apoptosis processes in MC3T3-E1 cells. Our findings suggested that zinc deficiency is capable of inducing apoptosis through a mitochondria-mediated pathway in osteoblastic cells.

From Local to Global

IN clinical practice, lipid emulsion is used commonly as a component of parenteral nutrition. Lipid emulsion also is used as therapy for severe cardiotoxicity secondary to accidental overdose of local anesthetics, an effect that has been confirmed in animals1–3and humans.4,5Because patients with local anesthetic-induced cardiac arrest are considered to be less responsive for standard resuscitation methods, this finding is striking, and currently infusion of lipid emulsion is considered the primary treatment for local anesthetic toxicity. In this issue of Anesthesiology, Rahman et al. 6report a new and potentially clinically relevant benefit of lipid emulsion therapy.Classic preconditioning7produces in the heart a state profoundly protected from ischemia–reperfusion injury; however, the prerequisite for knowing when the ischemic injury will occur has limited the clinical translation and relevance of preconditioning. Recently, a potentially more clinically relevant form of cardiac protection, termed postconditioning, has been described.8Postconditioning involves the application of protective interventions (e.g. , brief ischemia–reperfusion, volatile anesthetics, opioids, and other classic cardioprotective agents) after ischemia but before reperfusion.Rahman et al. extend the list of postconditioning agents to include Intralipid (Sigma, St. Louis, MO). They show that lipid emulsion infusion just before reperfusion (i.e. , postconditioning) protects from myocardial ischemia–reperfusion injury.9Subsequent mechanistic studies define a role for glycogen synthase kinase-3β and the mitochondrial permeability transition pore (mPTP) in this response. Mitochondria, a source of cellular adenosine triphosphate, are increasingly implicated in cell survival and death signaling in the heart. In particular, the mPTP has been suggested as the final effector in cardiac myocyte protection.10The mPTP opening in response to stress leads to an increase in mitochondrial membrane permeability to small molecules, resulting in cellular apoptosis and necrosis. Thus, regulating the mPTP opening has been considered to be a promising target for cardiac protection, and others have suggested the involvement of mPTP in postconditioning.11,12Postconditioning is mediated via a complex molecular signaling cascade involving the reperfusion injury salvage kinase and the survivor activating factor enhancement pathways.13Rahman et al. implicate common survival pathways, such as PI3K-Akt and ERK, in lipid emulsion-induced protection. In addition, they link these upstream survival kinases to downstream regulation of glycogen synthase kinase-3β. Recent work has shown that increased phosphorylation of glycogen synthase kinase-3β reduces the affinity of the adenine nucleotide translocase for cyclophilin D, suggesting that assembly of the complex is targeted by protective signals to limit mPTP opening.14Thus, there appears to be a direct link between survival kinase regulation and mPTP end effector function.These intriguing findings by Rahman et al. leave us with some important questions to be addressed in future investigation. First, how does lipid emulsion activate survival kinases—via a direct effect or secondary to receptor stimulation? General protective interventions appear to signal through G-protein–coupled receptor pathways,15including preconditioning and postconditioning via opioids, volatile anesthetics, and other cardioprotective agents. It would be important to determine whether lipid emulsion is sensitive to G-protein–coupled receptor inhibition. Second, does intra-lipid modulate membrane lipid composition to modulate cardiac protection? Signaling components involved in cardioprotection are localized in membrane microdomains (i.e. , lipid rafts and caveolae) that are enriched in cholesterol and sphingolipids.16,17A recent publication suggests there may be some link between lipid emulsion and caveolin18; however, a direct effect of lipid emulsion on caveolins was not tested in this study. Other studies show that perturbing caveolae can alter survival kinase signaling critical to protection from cardiac and neuronal ischemia–reperfusion injury.19–24It would be interesting to know whether Intralipid, being a lipid emulsion, affects membrane microdomains such as lipid rafts and caveolae to produce protection. Third, does the protective effect of lipid emulsion identified in this study play some role in the effects of propofol (where 10% lipid emulsion is the vehicle) that have been noted in ischemia–reperfusion injury? The potential protective effects of propofol are controversial,25,26and the lipid emulsion effects need to be considered separately from the potential direct effects of propofol. Studies using fospropofol (a water-soluble formulation of a phosphate ester prodrug of propofol) may be useful in resolving this issue.In summary, the current study by Rahman et al. focuses on a potentially important role for lipid emulsion in cardiac protection. Additional studies are needed to focus on mechanisms and potential clinical trials in humans. If similar protective effects are demonstrated in patients, lipid emulsion may play a novel and important role as a therapeutic intervention for patients with ischemic heart disease.* Department of Anesthesiology, University of California, San Diego, La Jolla, California. † Department of Anesthesiology, University of California, San Diego, and Veterans Affairs San Diego Healthcare System, San Diego, California. hepatel@ucsd.edu

Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-xL overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.

The Role of Mitochondrial Chaperone Tumor Necrosis Factor-associated Protein 1 (TRAP1) in the Regulation of Apoptosis

An increase of mitochondrial membrane permeability is one of the key events in apoptosis, since it leads to the release of mitochondrial apoptogenic factors, such as cytochrome c, into the cytoplasm that activate downstream target of apoptotic cell death. Bcl-2 family is one of the best-characterized proteins that directly regulate mitochondrial functions. A major role of the Bcl-2 family of proteins is to alter mitochondrial membrane permeability, thus controlling the release of caspase-activating cytochrome c. Recent reports describe about involvement of interesting apoptogenic regulators other than Bcl-2 family in regulation of mitochondrial function. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the heat-shock family of mitochondrial proteins, and substantially homologous to members of the 90-kDa families of heat-shock proteins (HSP90). TRAP1 seems to have specific functions differ from those of other members of the HSP90 family. Downregulation of TRAP1 expression enhances the release of cytochrome c from mitochondria. Moreover, reactive oxygen species (ROS) are involved in the regulation of the TRAP1 expression, indicating that TRAP1 is a sensor that involved in ROS mediated regulation of apoptosis. Here, we describe the mechanisms underlying the regulation of mitochondrial functions during apoptosis by TRAP1.

Effect ofMucuna prurienson oxidative stress mediated damage in aged rat sperm

Mucuna pruriens Linn., a leguminous plant, has been recognized as an aphrodisiac and spermatogenic agent. Protective efficacy of M. pruriens on reactive oxygen species (ROS)-induced pathophysiological alterations in structural and functional integrity of epididymal sperm in aged Wister albino rat was analysed. Animals were grouped as groups I, II, III and IV, i.e. young (control), aged, aged treated with ethanolic extract (200 mg/kg b.w.) of M. pruriens and young rats treated with M. pruriens, respectively. At the end of the experimental period, i.e. after 60 days animals were sacrificed, epididymal sperm were collected and subjected to count, viability, motility, morphology and morphometric analysis. Enzymatic and non-enzymatic antioxidants, ROS, lipid peroxidation (LPO), DNA damage, chromosomal integrity and mitochondrial membrane potential were estimated. Results obtained from the aged animals showed significant reduction in sperm count, viability and motility, increased morphological damage and an increase in the number of sperm with cytoplasmic remnant, and these alterations were significantly reversed in M. pruriens treated group. Significant increase in LPO, HO and H(2)O(2) production and significant decline in the levels of the enzymatic and non-enzymatic antioxidants were observed in the aged animals. Supplementation of M. pruriens significantly reduced ROS and LPO production and significant increase in both enzymatic and non-enzymatic antioxidant levels. There were significant DNA damage, loss of chromosomal integrity and increase in mitochondrial membrane permeability in aged rat sperm. This was significantly reduced in group III. Present observation indicates the antioxidant enhancing property, free radical quenching ability and spermatogenic efficacy of the M. pruriens. Collectively, sperm damage in ageing was significantly reduced by quenching ROS, improving antioxidant defence system and mitochondrial function.