Metformin induces both caspase-dependent and poly(ADP-ribose) polymerase-dependent cell death in breast cancer cells

There is substantial evidence that metformin, a drug used to treat type 2 diabetics, is potentially useful as a therapeutic agent for cancer. However, a better understanding of the molecular mechanisms through which metformin promotes cell-cycle arrest and cell death of cancer cells is necessary. It will also be important to understand how the response of tumor cells differs from normal cells and why some tumor cells are resistant to the effects of metformin. We have found that exposure to metformin induces cell death in all but one line, MDA-MB-231, in a panel of breast cancer cell lines. MCF10A nontransformed breast epithelial cells were resistant to the cytotoxic effects of metformin, even after extended exposure to the drug. In sensitive lines, cell death was mediated by both apoptosis and a caspase-independent mechanism. The caspase-independent pathway involves activation of poly(ADP-ribose) polymerase (PARP) and correlates with enhanced synthesis of PARP and nuclear translocation of apoptosis-inducing factor (AIF), which plays an important role in mediating cell death. Metformin-induced, PARP-dependent cell death is associated with a striking enlargement of mitochondria. Mitochondrial enlargement was observed in all sensitive breast cancer cell lines but not in nontransformed cells or resistant MDA-MB-231. Mitochondrial enlargement was prevented by inhibiting PARP activity or expression. A caspase inhibitor blocked metformin-induced apoptosis but did not affect PARP-dependent cell death or mitochondrial enlargement. Thus, metformin has cytotoxic effects on breast cancer cells through 2 independent pathways. These findings will be pertinent to efforts directed at using metformin or related compounds for cancer therapy.     

Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines

The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.     

DNA binding is required for the apoptogenic action of apoptosis inducing factor

The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.     

Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells

Taxol is extensively used clinically for chemotherapy of patients with ovarian, breast, and lung cancer. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. To determine the mechanism of action of taxol in ovarian cancer, we tested the effects of the drug, on the human ovarian carcinoma cell line, SKOV3. We observed that taxol-induced apoptosis of these cells by phosphatidylserine (PS) externalization and DNA fragmentation. While treatment of cells with taxol resulted in bcl-2 phosphorylation and mitochondrial depolarization, cytochrome c was not released and pro-caspase-3 was not activated. Treatment of SKOV3 cells with taxol, however, resulted in the translocation of AIF from the mitochondria to the nucleus via the cytosol. Taken together, these findings suggest that in SKOV3 cells, taxol induces caspase-independent AIF-dependent apoptosis.     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Mechanism of the malabaricone C-induced toxicity to the MCF-7 cell line

Abstract

In this study, we studied the mechanism of the cytotoxicity of malabaricone C (mal C) against human breast cancer MCF-7 cell line. Mal C dose-dependently increased the sub G1 cell population, associated with cytoplasmic oligonucleosome formation and chromatin condensation. The mal C-induced apoptosis led to mitochondrial damage as revealed by fluorescence microscopy and flow cytometry of the JC-1-stained cells as well as from the release of mitochondrion-specific nuclease proteins AIF and endo G. Mal C also released intracellular Ca²⁺ from the MCF-7 cells, but the Ca²⁺-modulators BAPTA-AM and Ru360 only partially abrogated the apoptosis. The calpain activation by mal C did not have any effect on its cytotoxicity. On the other hand, after mal C treatment significant lysosomal membrane permeabilization (LMP), along with release of cathepsin B, as well as Bid-cleavage and its translocation to mitochondria were observed much earlier than the mitochondrial damage. This suggested that cytotoxicity of mal C against human MCF-7 human breast cancer cell line may proceed through LMP as the initial event that triggered a caspase-independent, but cathepsin B and t-Bid-dependent intrinsic mitochondrial apoptotic pathway. A significant accumulation of cells in the S or G2-M phases along with upregulation of the cyclins E and A due to mal C exposure promises it to be a potential anti-cancer agent.     

Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.     

Transglutaminase 2 promotes both caspase-dependent and caspase-independent apoptotic cell death via the calpain/Bax protein signaling pathway

Transglutaminase 2 (TG2) is a versatile protein that is implicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse of ΔΨ(m), caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpain-induced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer.     

Selective targeting of breast cancer cells through ROS-mediated mechanisms potentiates the lethality of paclitaxel by a novel diterpene, gelomulide K

Defects in apoptotic pathways confer resistance to tubulin-binding agents via downregulation of caspases or overexpression of antiapoptotic factors, urging the need for novel agents acting on an alternative pathway. The purpose of this study was to investigate whether induction of ROS can induce caspase-independent cell death in breast cancer cells and thereby enhance the activity of paclitaxel. Here, we report that gelomulide K acts as a caspase-independent cell death-inducing agent that synergizes with paclitaxel in breast cancer cells and has low toxicity in normal cells. Treatment with gelomulide K induced PARP-1 hyperactivation, AIF nuclear translocation, and cytoprotective autophagy. These effects were associated with increased ROS production and a decrease in cellular GSH levels in cancer cells. Furthermore, pretreatment with NAC, a precursor of intracellular GSH, effectively abrogated gelomulide K-induced caspase-independent cell death and autophagy, suggesting that ROS-mediated downstream signaling is essential to the anticancer effects of gelomulide K. Additionally, in a xenograft model, gelomulide K induced PARP-1 activation and reduced tumor growth. In terms of structure-activity relationships, analysis not only showed a correlation between ROS levels and drug activity but also highlighted the importance of the 8,14-epoxy group. Taken together, our results show that enhancement of paclitaxel activity can be achieved with gelomulide K and that the structurally relevant pharmacophore provides important insight into the development of new caspase-independent cell death-inducing agents.     

Steroid receptor coactivator-interacting protein (SIP) inhibits caspase-independent apoptosis by preventing apoptosis-inducing factor (AIF) from being released from mitochondria

Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis.     

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.     

Dopaminochrome induces caspase-independent apoptosis in the mesencephalic cell line, MN9D

Parkinson's disease is characterized by a deficiency in motor cortex modulation due to degeneration of pigmented dopaminergic neurons of the substantia nigra projecting to the striatum. These neurons are particularly susceptible to oxidative stress, perhaps because of their dopaminergic nature. Like all catecholamines, dopamine is easily oxidized, first to a quinone intermediate and then to dopaminochrome (DAC), a 5-dihydroxyindole tautomer, that is cytotoxic in an oxidative stress-dependent manner. Here we show, using the murine mesencephalic cell line MN9D, that DAC causes cell death by apoptosis, illustrated by membrane blebbing, Annexin V, and propidium iodide labeling within 3 h. In addition, DAC causes oxidative damage to DNA within 3 h, and positive terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescence by 24 h. DAC, however, does not induce caspase 3 activation and its cytotoxic actions are not prevented by the pan-caspase inhibitor, Z-VAD-fmk. DAC-induced cytotoxicity is limited by the PARP1 inhibitor, 5-aminoisoquinolinone, supporting a role for apoptosis-inducing factor (AIF) in the apoptotic process. Indeed, AIF is detected in the nuclear fraction of MN9D cells 3 h after DAC exposure. Taken together these results demonstrate that DAC induces cytotoxicity in MN9D cells in a caspase-independent apoptotic manner, likely triggered by oxidative damage to DNA, and involving the translocation of AIF from the mitochondria to the nucleus.     

Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.     

α7 Nicotinic receptor activation reduces β-amyloid-induced apoptosis by inhibiting caspase-independent death through phosphatidylinositol 3-kinase signaling

The neurotoxicity of amyloid-β (Aβ) involves caspase-dependent and -independent programmed cell death. The latter is mediated by the nuclear translocation of the mitochondrial flavoprotein apoptosis inducing factor (AIF). Nicotine has been shown to decrease Aβ neurotoxicity via inhibition of caspase-dependent apoptosis, but it is unknown if its neuroprotection is mediated through caspase-independent pathways. In the present study, pre-treatment with nicotine in rat cortical neuronal culture markedly reduced Aβ(1-42) induced neuronal death. This effect was accompanied by a significant reduction of mitochondrial AIF release and its subsequent nuclear translocation as well as significant inhibition of cytochrome c release and caspase 3 activation. Pre-treatment with selective α7nicotinic acetylcholine receptor(nAChR) antagonist (methyllycaconitine), but not the α4 nAChR antagonist (dihydro-β-erythroidine), could prevent the neuroprotective effect of nicotine on AIF release/translocation, suggesting that nicotine inhibits the caspase-independent death pathway in a α7 nAChR-dependent fashion. Furthermore, the neuroprotective action of nicotine on AIF release/translocation was suppressed by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Pre-treatment with nicotine significantly restored Akt phosphorylation, an effector of PI3K, in Aβ(1-42) -treated neurons. These findings indicate that the α7 nAChR activation and PI3K/Akt transduction signaling contribute to the neuroprotective effects of nicotine against Aβ-induced cell death by modulating caspase-independent death pathways.     

Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose-dependent reduction in survivin protein. Although 74% of the SAO-treated MSN cells were trypan blue(+), PARP cleavage or activated caspase-3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co-administration of z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk) with SAO did not inhibit cell death suggesting a caspase-independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO-treated TC620 cells were trypan blue(+), PARP was cleaved, cells were TUNEL(+) and PI-staining revealed macronuclei and numerous apoptotic bodies. Co-treatment of the TC620 cells with SAO and zVAD-fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two-fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase-independent mechanism.     

The role of p38 MAPK and JNK in Arsenic trioxide-induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.     

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.     

Role for apoptosis-inducing factor in the physiological death of cerebellar neurons

Apoptosis-inducing factor (AIF) is implicated in caspase-independent apoptotic-like death. AIF released from mitochondria translocates to the nucleus, where it mediates some apoptotic events such as chromatin condensation and DNA degradation. Here, the role of AIF in the neuronal death was studied under physiological conditions. When we analyzed the cellular localization of AIF during cerebellar development, we found a significant increase in the number of neurons with nuclear AIF localization in an age-dependent manner. On the other hand, cerebellar granule neurons (CGN) chronically cultured in low concentration of potassium (5 mM; K5) die with apoptotic-like characteristics after five days. In the present study we found that K5 induces a caspase-dependent apoptotic-like death of CGN as well as a late nuclear translocation of AIF. When CGN death induced by K5 was carried out in the presence of a general inhibitor of caspases, there was a slight decrement of cell death, but neurons eventually died by showing apoptotic-like features such as phosphatidylserine translocation and nuclear condensation. Besides, there was a significant increment of nuclear AIF translocation. These findings support the idea that AIF could be involved in apoptotic-like death of CGN and that it could be an alternative mechanism of neuronal death during cerebellar development.     

α-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

α-Tomatine, a tomato glycoalkaloid, has been reported to possess antibiotic properties against human pathogens. However, the mechanism of its action against leukemia remains unclear. In this study, the therapeutic potential of α-tomatine against leukemic cells was evaluated in vitro and in vivo. Cell viability experiments showed that α-tomatine had significant cytotoxic effects on the human leukemia cancer cell lines HL60 and K562, and the cells were found to be in the Annexin V-positive/propidium iodide-negative phase of cell death. In addition, α-tomatine induced both HL60 and K562 cell apoptosis in a cell cycle- and caspase-independent manner. α-Tomatine exposure led to a loss of the mitochrondrial membrane potential, and this finding was consistent with that observed on activation of the Bak and Mcl-1 short form (Mcl-1s) proteins. Exposure to α-tomatine also triggered the release of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus and down-regulated survivin expression. Furthermore, α-tomatine significantly inhibited HL60 xenograft tumor growth without causing loss of body weight in severe combined immunodeficiency (SCID) mice. Immunohistochemical test showed that the reduced tumor growth in the α-tomatine-treated mice was a result of increased apoptosis, which was associated with increased translocation of AIF in the nucleus and decreased survivin expression ex vivo. These results suggest that α-tomatine may be a candidate for leukemia treatment.