Loss Of Mitochondrial Transmembrane Potential Research Articles

Identification of nanoscale ingredients in commercial food products and their induction of mitochondrially mediated cytotoxic effects on human mesenchymal stem cells.

Titanium dioxide (E171) and silicon dioxide (E551) are common additives found in food products, personal-care products, and many other consumer products used in daily life. Recent studies have reported that these food additives (manufactured E171 and E551) contain nanosized particles of less than 100 nm. However, the particle size distribution and morphology of added TiO2 and SiO2 particles are not typically stated on the package label. Furthermore, there is an increasing debate regarding health and safety concerns related to the use of synthetic food additives containing nanosized ingredients in consumer products. In this study, we identified the size and morphology of TiO2 and SiO2 particles in commercially available food products by using transmission electron microscope (TEM). In addition, the in vitro toxicological effects of E171 and E551 on human mesenchymal stem cells (hMSCs), an adult stem cell-based model, were assessed using the MTT assay and a flow cytometry-based JC-1 assay. Our TEM results confirmed the presence of nanoscale ingredients in food products, and the in vitro toxicology results indicated that the nanoscale E171 and E551 ingredients induced dose-dependent cytotoxicity, changes in cellular morphology, and the loss of mitochondrial trans-membrane potential in hMSCs. These preliminary results clearly demonstrated that the nanoscale E171 and E551 particles had adverse effects on hMSCs by inducing oxidative stress-mediated cell death. Accordingly, further studies are needed to identify the specific pathway involved, with an emphasis on differential gene expression in hMSCs.

Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells

A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation.

Enzymatic assays for probing mitochondrial apoptosis.

Isolated mitochondria are an invaluable analytical tool to probe mitochondrial function and evaluate apoptosis induction via the so-called mitochondrial pathway. Irrespective of their tissue origin (e.g., heart, liver, muscle, brain), these organelles participate actively to cell and life decision by producing energy for cell metabolism, but also by undergoing a lethal and irreversible mitochondrial membrane permeabilization (MMP) in stress and pathological conditions. MMP consequences consist, at least in part, in loss of mitochondrial transmembrane potential (ΔΨm), matrix swelling, arrest of respiration and ATP production, and cytochrome c release from the intermembrane space to the cytosol. These parameters can be evaluated in vitro via several miniaturized assays, which have tremendous applications in the field of pharmacology, toxicology, diagnosis, as well as drug discovery.

Britannin, a sesquiterpene lactone, inhibits proliferation and induces apoptosis through the mitochondrial signaling pathway in human breast cancer cells.

Induction of apoptosis in cancer cells can be a promising treatment method in cancer therapy. Naturally derived products had drawn growing attention as agent in cancer therapy. The main target of anticancer drugs may be distinct, but eventually, they lead to identical cell death pathway, which is apoptosis. Here, we indicated that britannin, a sesquiterpene lactone isolated from Asteraceae family, has antiproliferative activity on the MCF-7 and MDA-MB-468 human breast cancer cells. Annexin V/propidium iodide (PI) staining, Hoechst 33258 staining, and caspase-3/9 activity assay confirmed that britannin is able to induce apoptosis in MCF-7 and MDA-MB-468 cells. The Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to britannin treatment, while the expression of Bax protein was increased, which were positively correlated with elevated expression of p53. Moreover, britannin also increased reactive oxygen species (ROS) generation which in turn triggered the loss of mitochondrial transmembrane potential (ΔΨm) and the subsequent release of cytochrome c from mitochondria into cytosol. Taken together, these results suggest that britannin inhibits growth of MCF-7 and MDA-MB-468 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may potentially serve as an agent for breast cancer therapy.

A polysaccharide from Sanguisorbae radix induces caspase-dependent apoptosis in human leukemia HL-60 cells

One polysaccharide (SRP) was purified from Sanguisorbae radix by DEAE-cellulose-52 anion-exchange and Sephacryl S-400 gel filtration chromatography. The aim of this study was to evaluate the anticancer efficacy of SRP on human leukemia HL-60 cells in vitro and unveil the underlying mechanisms. Our results showed that SRP was able to suppress the proliferation of HL-60 cells in a dose-dependent manner by the mechanism involved in the induction of apoptosis. The increase in SRP-induced apoptosis was correlated with a rapid and sustained loss of mitochondrial transmembrane potential (ΔΨm) and a release of cytochrome c from the mitochondria into the cytosol. Furthermore, Western blot and RT-PCR analysis revealed that the protein and mRNA levels of antiapoptotic Bcl-2 were downregulated, whereas those of pro-apoptotic Bax were upregulated. Besides, caspase-9 and caspase-3 were activated, while caspase-8 was intact. Additionally, the apoptotic cells by SRP were significantly inhibited by a caspase-3 inhibitor (z-DEVD-fmk) or a caspase-9 inhibitor (Z-LETD-FMK), demonstrating the important role of caspase-9 and -3 in the process. Taken together, these findings provided evidence that SRP induced the apoptosis of HL-60 cells through an intrinsic mitochondria-mediated signaling pathway and SRP may be a promising chemotherapeutic agent for treatment of leukemia.

Emodin-Provoked Oxidative Stress Induces Apoptosis in Human Colon Cancer HCT116 Cells through a p53-Mitochondrial Apoptotic Pathway

Emodin, a natural anthraquinone isolated from the traditional Chinese medicine Radix rhizoma Rhei, can induce apoptosis in many kinds of cancer cells. This study demonstrated that emodin induces apoptosis in human colon cancer HCT116 cells by provoking oxidative stress, which subsequently triggers a p53-mitochondrial apoptotic pathway. Emodin induced mitochondrial transmembrane potential loss, increase in Bax and decrease in Bcl-2 expression and mitochondrial translocation and release of cytochrome c to cytosol in HCT116 cells. In response to emodin-treatment, ROS increased rapidly, and subsequently p53 was overexpressed. Pretreatment with the antioxidant NAC diminished apoptosis and p53 overexpression induced by emodin. Transfecting p53 siRNA also attenuated apoptosis induced by emodin, Bax expression and mitochondrial translocation being reduced compared to treatment with emodin alone. Taken together, these results indicate that ROS is a trigger of emodin-induced apoptosis in HCT116 cells, and p53 expression increases under oxidative stress, leading to Bax-mediated mitochondrial apoptosis.

The ERK/eIF4F/Bcl-XL pathway mediates SGP-2 induced osteosarcoma cells apoptosis in vitro and in vivo

The aim of this study is to assess the molecular foundation of anti-tumor activity of SGP-2 in osteosarcoma cells. SGP-2 significantly blocks cell proliferation in human osteosarcoma U2OS cell model and inhibits tumor growth without causing apparent toxicity effect in mouse sarcoma S-180 cell-derived tumor model. Moreover, SGP-2 induces intrinsic apoptosis including the activation of caspase-3/7/9, the loss of mitochondrial transmembrane potential (ΔΨm), and the release of cytochrome c from mitochondrion, controlled by the down-regulation of B-cell lymphoma-extra large (Bcl-XL). Further research reveals that SGP-2 inhibits the assembly of eukaryotic initiation factor 4F (eIF4F) complex which is responsible for the decline of Bcl-XL. Finally, extracellular-signal-regulated kinase (ERK) controls SGP-2 induced intrinsic apoptosis. Taken together, SGP-2 exerts anti-tumor effect through intrinsic apoptotic pathway controlled by ERK/eIF4F/Bcl-XL pathway.

Changes in mitochondrial function induced by dequalinium precede oxidative stress and apoptosis in the human prostate-cancer cell line PC-3

Mitochondria play central roles in diverse physiological and pathological conditions associated with cell survival and death. Delocalized lipophilic cations, such as dequalinium (DQA), are accumulated in cancer cells attracted by the highly negative mitochondrial transmembrane potential of these cells. DQA showed a potent anticancer activity in cells from different malignancies. Here, we report the effect of DQA on PC-3 prostate cancer cells. Incubation with DQA at concentrations between 1.5 and 100 microM from 24 to 48 h decreases cell viability. The decrease in cell viability together with a loss of mitochondrial transmembrane potential induced an increase in reactive oxygen species production and cell death via caspase-3 dependent apoptotic pathway. QA was shown to cause moderate to strong cell death in a time and concentration dependent manner, causing a most advantageous effect at a concentration of 10 microM applied for a long 48 h time period, which might be a consequence of the kinetics of intracellular DQA accumulation in mitochondria, but also of the mechanisms of DQA-induced cell death. This data shows DQA as a promising agent against the human prostate cancer PC-3 cell line, activating the caspase-3 dependent apoptotic pathway. This fact might be beneficial for possible future applications in cancer therapy.

Cranberry extract initiates apoptosis in HL‐60 cell line through intrinsic apoptotic pathway (644.9)

The potential anticancer activity of cranberry was reported for the first time in 1996. Proanthocyandins (PACs), the most abundant and complex flavonoid in cranberry, have been described as the major contributors of the anticancer activity reported in the literature. One of the proposed chemopreventative mechanisms is the induction of apoptosis by cranberry PACs. However, limited studies have been done to investigate the role of cranberry in initiating apoptosis in cancer cells and its underlying mechanism of action remains undetermined. Therefore, the overall aim of this study was to investigate the apoptotic pathway that cranberry affects, intrinsic vs. extrinsic pathway. In our study, HL‐60 cells were incubated with 25 µg/ml cranberry‐(60‐65%)PAC extract for 24 hours. Cranberry treatment showed an 85 % increase in caspase 3/7 activity. The data also showed a great enhancement in the activation of caspase‐9 (379 %), but it did not exhibit any change in caspase‐8 activity. Further tests revealed that cranberry treatment caused a loss of mitochondrial transmembrane potential leading to an increase in the mitochondrial release of cytochrome C (87.5%) and Smac (41%). These findings indicate that this cranberry‐PAC extract can induce apoptosis in HL‐60 cells through triggering the intrinsic (mitochondrial) apoptotic pathway.Grant Funding Source: Funded by Agricultural Experiment Station and Ocean Spray Cranberries, Inc.

A novel protein extracted from foxtail millet bran displays anti-carcinogenic effects in human colon cancer cells

Millet is an important cereal food and exhibits multiple biological activities, including immunodulatory, antioxidant, antifungal and anti-hyperglycemia effects. Herein, we describe a novel 35kDa protein with anti-cancer properties, named FMBP, which was extracted and purified from foxtail millet bran by cell-based screening. FMBP is highly homologous to peroxidase as revealed by mass spectrometry and gene sequencing analysis. Furthermore, in vivo anti-tumor results implicated that the novel protein FMBP had the ability to suppress xenografted tumor growth in nude mice. Mechanistically, FMBP is able to suppress colon cancer cell growth through induction of G1 phase arrest and also causes a loss of mitochondrial transmembrane potential which results in caspase-dependent apoptosis in colon cancer cells. Notably, FMBP has much lower toxicity in normal colon epithelial cells. Taken together, FMBP has targeted anti-colon cancer effects and may serve as a therapeutic agent against colon cancer.

Rosamines Targeting the Cancer Oxidative Phosphorylation Pathway

Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM), inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = −7 (GI50 = 0.1 µM) and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6) exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

Triptolide induces apoptotic cell death of multiple myeloma cells via transcriptional repression of Mcl-1

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Levocarnitine protects H9c2 rat cardiomyocytes from H2O2-induced mitochondrial dysfunction and apoptosis.

Background: Although the protective effects of levocarnitine in patients with ischemic heart disease are related to the attenuation of oxidative stress injury, the exact mechanisms involved have yet to be fully understood. Our aim was to investigate the potential protective effects of levocarnitine pretreatment against oxidative stress in rat H9c2 cardiomyocytes.Methods: Cardiomyocytes were exposed to H2O2 to create an oxidative stress model. The cells were pretreated with 50, 100, or 200 μM levocarnitine for 1 hour before H2O2 exposure.Results: H2O2 exposure led to significant activation of oxidative stress in the cells, characterized by reduced viability, increased intracellular reactive oxygen species, lipid peroxidation, and reduced intracellular antioxidant activity. Mitochondrial dysfunction was also observed following H2O2 exposure, reflected by the loss of mitochondrial transmembrane potential and intracellular adenosine triphosphate. These pathophysiological processes led to cardiomyocyte apoptosis through activation of the intrinsic apoptotic pathway. More importantly, the levocarnitine pretreatment attenuated the H2O2-induced oxidative injury significantly, preserved mitochondrial function, and partially prevented cardiomyocyte apoptosis during the oxidative stress reaction. Western blotting analyses suggested that levocarnitine pretreatment increased plasma protein levels of Bcl-2, reduced Bax, and attenuated cytochrome C leakage from the mitochondria in the cells.Conclusion: Our in vitro study indicated that levocarnitine pretreatment may protect cardiomyocytes from oxidative stress-related damage.

Response to rotenone is glucose-sensitive in a model of human acute lymphoblastic leukemia: involvement of oxidative stress mechanism, DJ-1, Parkin, and PINK-1 proteins.

To establish the effect of low (11 mM) and high (55 mM) glucose concentrations (G11, G55) on Jurkat cells exposed to rotenone (ROT, a class 5 mitocan). We demonstrated that ROT induces apoptosis in Jurkat cells cultured in G11 by oxidative stress (OS) mechanism involving the generation of anion superoxide radical (O2 ∙−, 68%)/hydrogen peroxide (H2O2, 54%), activation of NF-κB (32%), p53 (25%), c-Jun (17%) transcription factors, and caspase-3 (28%), apoptosis-inducing factor (AIF, 36%) nuclei translocation, c-Jun N-terminal kinase (JNK) activation, and loss of mitochondria transmembrane potential (ΔΨm, 62%) leading to nuclei fragmentation (~10% and ~40% stage I-II fragmented nuclei, resp.). ROT induces massive cytoplasmic aggregates of DJ-1 (93%), and upregulation of Parkin compared to untreated cells, but no effect on PINK-1 protein was observed. Cell death marker detection and DJ-1 and Parkin expression were significantly reduced when cells were cultured in G55 plus ROT. Remarkably, metformin sensitized Jurkat cells against ROT in G55. Our results indicate that a high-glucose milieu promotes resistance against ROT/H2O2-induced apoptosis in Jurkat cells. Our data suggest that combined therapy by using mitochondria-targeted damaging compounds and regulation of glucose (e.g., metformin) can efficiently terminate leukemia cells via apoptosis in hyperglycemic conditions.

Administration Dependent Antioxidant Effect of Carica papaya Seeds Water Extract.

Carica papaya is widely used in folk medicine as herbal remedy to prevent, protect against, and cure several diseases. These curative properties are based on the presence in different parts of the plant of phytochemical nutrients with antioxidant effect. Seeds are the less exploited part; thus this study is aimed at assessing the antioxidant activities of the C. papaya seeds water extract against hydrogen peroxide (H2O2) oxidative stress in human skin Detroit 550 fibroblasts. C. papaya seeds water extract is not toxic and acts as a potent free radical scavenger, providing protection to Detroit 550 fibroblasts that underwent H2O2 oxidative stress. Data show that (i) the maximum protective effect is achieved by the simultaneous administration of the extract with 1 mM H2O2; (ii) the extract in presence of an oxidative stress does not increase catalase activity and prevents the release of cytochrome C and the inner mitochondrial transmembrane potential (Δψ m) loss; (iii) the extract is more efficient than vitamin C to hamper the oxidative damage; (iv) the purified subfractions of the seeds water extract exert the same antioxidant effect of whole extract. In conclusion, C. papaya seeds water extract is potentially useful for protection against oxidative stress.

Phenethyl isothiocyanate induces calcium mobilization and mitochondrial cell death pathway in cholangiocarcinoma KKU-M214 cells

BackgroundPhenethyl isothiocyanate (PEITC) is a cancer chemopreventive agent from cruciferous vegetables. Cholangiocarcinoma (CCA) is a chemo-resistant cancer with very poor prognosis. We evaluated the effects of PEITC on induction of apoptotic cell death in relation to cellular glutathione (GSH) and mitochondrial function of a CCA cell line, KKU-M214.MethodsCytotoxic effects of PEITC on a CCA cell line, KKU-M214, and a reference cell line, Chang cells were evaluated. To delineate mechanisms of cell death, the following parameters were measured; GSH and superoxide levels as the oxidative status parameters, apoptosis related proteins levels using Western blotting. Cellular free calcium level and mitochondrial transmembrane potential were also measured.ResultsPEITC induced apoptotic cell death of both KKU-M214 and Chang cells. After PEITC treatment, both cells showed decrease of Bcl-xl and increase of Bax levels. While KKU-M214 cells released AIF, Chang cells released cytochrome c, with subsequent activation of caspase 3 and 9, upon PEITC treatment. PEITC induced superoxide formation in both cells, although it seemed not play a role in cell death. PEITC caused GSH redox stress in different ways in two cell types, because N-acetylcysteine (NAC) prevented redox stress in Chang but not in KKU-M214 cells. The loss of mitochondrial transmembrane potential was induced by PEITC concurrent with GSH stress, but was not a primary cause of cell death. The rapid increase of free calcium level in cytosol was associated with cell death in both cell lines. These events were prevented by NAC in Chang cells, but not in KKU-M214 cells.ConclusionPEITC induced cell death KKU-M214 cells and Chang cells via increase of cellular calcium mobilization and activation of mitochondrial cell death pathway. The effects of PEITC on the redox stress was mediated via different ways in CCA and Chang cells because NAC could prevent redox stress in Chang cells, but not in KKU-M214 cells. The multiple effects of PEITC may be useful for the development of novel chemotherapy for CCA.

Cytotoxic and Apoptotic-inducing Effects of Purple Rice Extracts and Chemotherapeutic Drugs on Human Cancer Cell Lines

Pigmented rice is mainly black, red, and dark purple, and contains a variety of flavones, tannin, polyphenols, sterols, tocopherols, γ-oryzanols, amino acids, and essential oils. The present study evaluated the cytotoxic effects of purple rice extracts (PREs) combined with chemotherapeutic drugs on human cancer cells and mechanisms of cell death. Methanolic (MeOH) and dichloromethane (DCM) extracts of three cultivars of purple rice in Thailand: Doisaket (DSK), Nan and Payao (PYO), were tested and compared with white rice (KK6). Cytotoxicity was determined by 3-(4, 5-dimethyl)-2, 5-diphenyltetrazolium bromide (MTT) assay in human hepatocellular carcinoma HepG2, prostate cancer LNCaP and murine normal fibroblast NIH3T3 cells. MeOH-PYO-PRE was the most cytotoxic and inhibited HepG2 cell growth more than that of LNCaP cells but was not toxic to NIH3T3 cells. When PREs were combined with paclitaxel or vinblastine, they showed additive cytotoxic effects on HepG2 and LNCaP cells, except for MeOH-PYO-PRE which showed synergistic effects on HepG2 cells when combined with vinblastine. MeOH-PYO-PRE plus vinblastine induced HepG2 cell apoptosis with loss of mitochondrial transmembrane potential (MTP) but no ROS production. MeOH-PYO-PRE-treated HepG2 cells underwent apoptosis via caspase-9 and-3 activation. The level of γ-oryzanol was highest in DCM-PYO-PRE (44.17 mg/g) whereas anthocyanin content was high in MeOH-PYO-PRE (5.80 mg/g). In conclusion, methanolic Payao purple rice extract was mostly toxic to human HepG2 cells and synergistically enhanced the cytotoxicity of vinblastine. Human HepG2 cell apoptosis induced by MeOH-PYO-PRE and vinblastine was mediated through a mitochondrial pathway.

The activation of p38 and JNK by ROS, contribute to OLO-2-mediated intrinsic apoptosis in human hepatocellular carcinoma cells

In this study, we describe that a novel synthesized compound, olean-28,13β-olide 2 (OLO-2), exhibits selective cytotoxic activity via inducing apoptosis in human hepatocellular carcinoma (HCC) cell lines but not normal human hepatic cells in vitro. Exposure of human HCC HepG2 cells to OLO-2 results in significant loss of mitochondrial transmembrane potential (ΔΨm), the release of cytochrome c, the recruitment of B-cell lymphoma 2 (Bcl-2) assaciated X protein (Bax) and the downregulation of Bcl-2. The apoptosis induced by OLO-2 is associated with the activation of caspase-3/9 and the nuclear translocation of apoptosis inducing factor (AIF). Moreover, the increase of phosphorylated p38 and c-Jun N-terminal kinase (JNK) is observed. OLO-2-induced the externalization of phosphatidyl-serine (PS) and the loss of ΔΨm are blocked by p38 inhibitor SB203580 or JNK inhibitor SP600125. In addition, OLO-2 provokes the generation of reactive oxygen species (ROS) in HepG2 cells, while the antioxidant N-acetyl cysteine (NAC) almost completely blocks OLO-2-induced apoptosis and the activation of p38 and JNK. Taken together, the present study demonstrates that OLO-2 exhibits its cytotoxic activity through intrinsic apoptosis via ROS generation and the activation of p38 and JNK. Its potential to be a candidate of anti-cancer agent is worth being further investigated.

The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance

Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.

Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

BackgroundComplementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcerea carbonica-induced tumor regression.MethodsTo investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells.ResultsInterestingly, although calcarea carbonica administration to Ehrlich’s ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. Knocking out of p53 by RNA-interference inhibited calcarea carbonica-induced apoptosis thereby confirming the contribution of p53.ConclusionThese observations delineate the significance of immuno-modulatory circuit during calcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.