Mitochondrial Caspase Pathway Research Articles

Arsenic trioxide induces proteasome dependent TBLR1-RARα degradation to improve leukemia eradication through cell differentiation enhancement.

Background: Acute promyelocytic leukemia (APL) mainly harbors PML-RARα fusion gene, which is sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) treatment. However, APL harboring other RARα fusion genes exhibit different drug sensitivity. Here, we investigated the role and mechanism of TBLR1-RARα, a rare RARα fusion gene, on ATO treatment in leukemia cells.Methods: By constructing two cell models of leukemia cell line HL-60 and U937 with overexpressed TBLR1-RARα, we detected the cell differentiation in the two cell models after ATO treatment by flow cytometry and Wright staining. Meanwhile, cell viability, colony formation and apoptosis were also determined after ATO treatment.Results: We found that TBLR1-RARα enhanced ATO-induced apoptosis and cell proliferation inhibition. Besides, TBLR1-RARα also promoted ATO-induced cell differentiation. Furthermore, we found that the mitochondrial caspase pathway was involved in the apoptosis induced by ATO treatment in TBLR1-RARα positive leukemia cells. Moreover, ATO mediated TBLR1-RARα protein degradation via proteasome pathway, which accounts for the transcriptional activation of RARα target gene and is further involved in cell differentiation of TBLR1-RARα positive leukemia cells.Conclusions: Our study provides evidence that TBLR1-RARα positive APL patients may benefit from ATO treatment, thereby improving the appropriate management in TBLR1-RARα positive APL.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Simple SummaryDiffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma with a high curability rate. However, 40% of patients will relapse or exhibit refractory disease, and compromised apoptotic pathways is among the prognosis-worsening factors. Therefore, drugging non-apoptotic modalities might be therapeutically promising. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells. Herein, we show that TQ selectively kills DLBCL cells, either cell lines or primary lymphoma cells bearing resistance features to standard treatment. Investigations show that, although TQ induced apoptotic markers, non-apoptotic death was the major mechanism responsible for TQ-induced cellular demise. We demonstrate critical and selective roles of cytosolic calcium and necroptosis in TQ-induced non-apoptotic cell death. Finally, TQ exhibits an improved selectivity profile over conventional chemotherapy. Collectively, this work provides new insights into the mode of action of TQ and points to the therapeutic relevance of non-apoptotic modalities as a fail-safe mechanism for pro-apoptotic DLBCL therapies.Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Methylene Blue-Mediated Photodynamic Therapy Induces Macrophage Apoptosis via ROS and Reduces Bone Resorption in Periodontitis.

Aim To investigate whether methylene blue-mediated photodynamic therapy (MB-PDT) can affect the “fate” of macrophages in vitro or in periodontitis tissues and to explore the potential mechanism. Methods For in vitro treatments, THP-1 macrophages were divided into three experimental groups: C/control, no treatment; MB, methylene blue treatment; and MB-PDT, MB and laser irradiation treatment. Then, apoptosis and apoptosis-related proteins were detected in each group. For in vivo treatments, periodontitis was ligature-induced in the first molars of the bilateral maxilla in 12 Sprague Dawley (SD) rats. After six weeks, the ligatures were removed and all the induced molars underwent scaling and root planning (SRP). Then, the rats were divided into three groups according to the following treatments: SRP, saline solution; MB, phenothiazinium dye; and MB-PDT, MB and laser irradiation. Apoptotic macrophages, inflammation levels, and alveolar bone resorption in the periodontal tissues of rats were analyzed in each group. Results In vitro, flow cytometry analysis demonstrated that 10 μM MB and 40 J/cm2 laser irradiation maximized the apoptosis rate (34.74%) in macrophages. Fluorescence probe and Western blot analyses showed that MB-PDT induced macrophage apoptosis via reactive oxygen species (ROS) and the mitochondrial-dependent apoptotic pathway. Conversely, the addition of exogenous antioxidant glutathione (GSH) and the pan-caspase inhibitor Z-VAD-FMK markedly reduced the apoptotic response in macrophages. In vivo, immunohistochemistry, histology, radiographic, and molecular biology experiments revealed fewer infiltrated macrophages, less bone loss, and lower IL-1β and TNF-α levels in the MB-PDT group than in the SRP and MB groups (P < 0.05). Immunohistochemistry analysis also detected apoptotic macrophages in the MB-PDT group. Conclusion MB-PDT could induce macrophage apoptosis in vitro and in rats with periodontitis. This may be another way for MB-PDT to relieve periodontitis in addition to its antimicrobial effect. Meanwhile, MB-PDT induced apoptosis in THP-1 macrophages via the mitochondrial caspase pathway.

Sonodynamic therapy-induced foam cells apoptosis activates the phagocytic PPARγ-LXRα-ABCA1/ABCG1 pathway and promotes cholesterol efflux in advanced plaque.

In advanced atherosclerotic plaques, defective efferocytosis of apoptotic foam cells and decreased cholesterol efflux contribute to lesion progression. In our previous study, we demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (SDT) could induce foam cells apoptosis via the mitochondrial-caspase pathway. In the current research, we sought to explore ALA-SDT-induced apoptosis of phagocytes and the effects of cholesterol efflux and efferocytosis in advanced apoE-/- mice plaque.Methods: apoE-/- mice fed western diet were treated with ALA-SDT and sacrificed at day 1, day 3, day 7 and day 28 post treatment. THP-1 macrophage-derived foam cells were treated with ALA-SDT. 5 hours later, the supernatant was collected and added to fresh foam cells (phagocytes). Then, the lipid area, efferocytosis, cholesterol efflux, anti-inflammatory reactions and PPARγ-LXRα-ABCA1/ABCG1 pathway were detected in plaque in vivo and in phagocytes in vitro.Results: We found that ALA-SDT induced foam cells apoptosis coupled with efferocytosis and upregulation of Mer tyrosine kinase (MerTK) both in vivo and in vitro. The lipid content in plaque decreased as early as 1 day after ALA-SDT and this tendency persisted until 28 days. The enhancement of phagocytes cholesterol efflux was accompanied by an approximately 40% decrease in free cholesterol and a 24% decrease in total cholesterol in vitro. More importantly, anti-inflammatory factors such as TGFβ and IL-10 were upregulated by ALA-SDT treatment. Finally, we found that PPARγ-LXRα-ABCA1/ABCG1 pathway was activated both in vivo and in vitro by ALA-SDT, which could be blocked by PPARγ siRNA.Conclusions: Activation of PPARγ-LXRα-ABCA1/ABCG1 pathway induced by ALA-SDT treatment engages a virtuous cycle that enhances efferocytosis, cholesterol efflux and anti-inflammatory reactions in advanced plaque in vivo and in phagocytes in vitro.

Polyphenol epigallocatechin-3-gallate inhibits hypoxia/reoxygenation-induced H9C2 cell apoptosis.

We aimed to observe the protective effect of EGCG on hypoxia/reoxygenation injury of H9C2 myocardial cells and to study the inhibition mechanism of EGCG on hypoxia/reoxygenation injury of H9C2 myocardial cells. H9C2 cells were used as the objects of study and hypoxia/reoxygenation cells were pretreated with EGCG in different concentrations. MTT, Hoechst 33258 and LDH were used to detect the viability and apoptosis of H9C2 cells. The protection mechanism of polyphenol epigallocatechin-3-gallate was studied via western blotting and mitochondrial membrane potential detection. Polyphenol epigallocatechin-3-gallate (10 μM) reduced the proportion of H9C2 cell apoptosis after hypoxia/reoxygenation (4/20 h), stabilized the mitochondrial membrane potential and decreased the expressions of mitochondrial damage-related proteins, thus protecting the mitochondrial function. Polyphenol epigallocatechin-3-gallate alleviates the hypoxia-/reoxygenation-induced H9C2 cell apoptosis, which may play the myocardial protective effect through inhibiting the mitochondrial Caspase pathway.

Ultrasound hyperthermia induces apoptosis in head and neck squamous cell carcinoma: an in vitro study

BackgroundHyperthermia is considered an efficient complement in the treatment of head and neck squamous cell carcinoma (HNSCC). Hyperthermia induces cell apoptosis in a temperature- and time-dependent manner. However, the molecular mechanism of hyperthermia remains unclear. The aim of this study was to investigate the mechanism of apoptosis induced by ultrasound hyperthermia in HNSCC cell lines HN-30 and HN-13.Material and MethodsWe examined the dynamic changes of early apoptosis and secondary necrosis in HN-30 and HN-13 cells treated by hyperthermia at 42°C for 10 min. We further examined mitochondrial membrane potential in vitro by ultrasound hyperthermia for different heating temperatures (38-44°C, 10 min) and heating times (42°C, 10-50 min). After heating by ultrasound at 42°C for 10 min, the apoptosis index achieved its highest level at 8 h after treatment, decreased rapidly and remained constant at a reduced level at 12 h.ResultsThe level of secondary necrosis increased with the level of early apoptosis but remained at a higher level until 14 h. The level of secondary necrosis correlated with the level of early apoptosis (HN-13: r=0.7523, P=0.0030; HN-30: r=0.6510, P=0.016). The fractions of cells with low mitochondrial membrane potential (Δψ) in the heating-temperature grads group and heating-time grads group decreased significantly over time. Therefore, HN-30 and HN-13 cells developed apoptosis after ultrasound hyperthermia treatment with decreases in the mitochondrial transmembrane potential level.ConclusionsUltrasound hyperthermia induces apoptosis in HN-30 and HN-13 cells, possibly via the mitochondrial caspase pathway. Key words:Head and neck squamous cell carcinoma, apoptosis, mitochondrial membrane potential, ultrasound hyperthermia.

Enhancement of anti-tumor effects of 5-fluorouracil on hepatocellular carcinoma by low-intensity ultrasound.

BackgroundHepatocellular carcinoma (HCC) accounts for 75 % of liver cancers and is the second most lethal cancer, associated with its multiple etiologies, poor prognosis and resistance to chemotherapy drugs. Chemotherapy treatment on HCC suffers low efficacy of drug uptake and can produce a range of side effects. Here we report an investigation on the effect of a combined treatment on human hepatocellular carcinoma BEL-7402 cells using low-intensity ultrasound (US) and 5-fluorouracil (5-FU).MethodsThe uptake of 5-FU was measured by the high-performance liquid chromatography (HPLC). DNA damage was detected by the comet assay. MTT assay was used to examine cell viability. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were respectively detected by the fluorescent probes DCFH-DA or JC-1. Endogenous apoptosis-associated proteins were analyzed by the western blot and immunohistochemistry. Histopathological changes were evaluated by the hematoxylin and eosin (H&E) staining. Cell apoptosis was evaluated by the TUNEL and flow cytometry assays. Cell proliferation was measured using the immunohistochemical staining of PCNA.ResultsOur results showed that low-intensity US (1.1 MHz, 1.0 W/cm2, 10 % duty cycle) significantly enhanced the uptake of 5-FU, 5-FU-mediated DNA damage and reactive oxygen species (ROS) generation. The increased ROS production up-regulated the p53 protein level, which led to the up-regulation of Bax and down-regulation of Bcl-2. The enhancement of ROS generation and the activation of the apoptosis-associated proteins further triggered the collapse of mitochondrial membrane potential, released cytochrome c from mitochondria into cytosol and activated the mitochondria-caspase pathway, and cell apoptosis. Such enhanced effects could be partially blocked by the ROS scavenger N-acetylcysteine (NAC). Overall, low-intensity US combined with 5-FU led to an effective inhibition of tumor growth and prolonged overall survival of BEL-7402 HCC-bearing nude mice by more than 15 % compared with 5-FU treatment alone.ConclusionsOur results showed that low-intensity ultrasound combined with 5-FU produced much enhanced synergistic anti-tumor effects via enhanced ROS production in treating HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0349-4) contains supplementary material, which is available to authorized users.

Apoptosis of THP-1 Macrophages Induced by Pseudohypericin-Mediated Sonodynamic Therapy Through the Mitochondria-Caspase Pathway

Background/Aims: Pseudohypericin (P-HY) and its congener hypericin are the major hydroxylated phenanthroperylenediones present in Hypericum species. Our previous study indicated that hypericin was able to function as a sonosensitizer. The potential use of P-HY as a sonosensitizer for sonodynamic therapy (SDT) requires further exploration. Thus, this study aimed to investigate the effects of P-HY-SDT on THP-1 macrophages. Methods: THP-1 macrophages were incubated with P-HY, and cell viability was measured using a CCK-8 assay. Fluorescence microscopy assessed the intracellular reactive oxygen species (ROS), mitochondrial membrane potential (∆Ψm) and mitochondrial permeability transition pore (mPTP) opening. Apoptotic and necrotic cell levels were measured by the flow cytometry analysis. Western blots were employed to assay BAX, Cytochrome C expression and apoptosis-related proteins. Results: P-HY-SDT induced THP-1 macrophage apoptosis. The levels of ROS were significantly increased in the SDT group, resulting in both mPTP opening and ∆Ψm loss, which led to apoptosis. In addition, the translocation of BAX, release of Cytochrome C and the upregulated expression of apoptosis-related proteins after P-HY-SDT were observed, all of which were reversed by N-acetyl cysteine (NAC). Conclusion: P-HY-SDT induced THP-1 macrophage apoptosis through the mitochondria-caspase pathway via ROS generation, the translocation of BAX and the release of Cytochrome C to regulate the mPTP opening.

Combination of Hydroxyl Acetylated Curcumin and Ultrasound Induces Macrophage Autophagy with Anti-Apoptotic and Anti-Lipid Aggregation Effects

Background/Aims: Sonodynamic therapy (SDT) is considered a new approach for the treatment of atherosclerosis. We previously confirmed that hydroxyl acetylated curcumin (HAC) was a sonosensitizer. In this study, we investigated the mechanism of THP-1 macrophage apoptosis and autophagy induced by HAC mediated SDT (HAC-SDT). Methods: Cell viability was measured using a CCK-8 assay. Laser scanning confocal microscopy was used to measure the levels of intracellular reactive oxygen species (ROS), sub-cellular HAC localization, BAX and cytochrome C translocation, LC3 expression, monodansylcadaverine staining and Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL) uptake. Flow cytometry was used to analyze apoptosis and autophagy via Annexin V/propidium iodide and acridine orange staining, respectively. The expression levels of apoptosis- and autophagy-related proteins were detected by Western blot. Oil red O was used to measure intracellular lipid accumulation. Results: We identified HAC (5.0 μg/mL) located in lysosomes, endoplasmic reticulum, Golgi apparatus and mitochondria after 4 h of incubation. Compared with other sonosensitizers (e.g., curcumin and emodin), HAC had a more obvious sonodynamic effect on macrophages. Furthermore, the mitochondrial-caspase pathway was confirmed to play a crucial role in the HAC-SDT-induced apoptosis; BAX translocated from the cytosol to the mitochondria during HAC-SDT. Subsequently, mitochondrial cytochrome C was released into the cytosol, activating the caspase cascade in a time-dependent manner. Furthermore, HAC-SDT could induce PI3K/AKT/mTOR pathway dependent autophagy, accompanied by a decrease in the lipid uptake of THP-1 macrophages. This mechanism was demonstrated by the formation of acidic vesicular organelles, the conversion of LC3 I to LC3 II, the expression of related proteins, and the attenuation of both Dil-ox-LDL and oil red O staining. Moreover, pre-treatment with the autophagy inhibitor 3-methyladenine enhanced the HAC-SDT-induced apoptosis. Additionally, HAC-SDT-induced autophagy and apoptosis were both blocked by ROS scavenger N-acetyl-l-cysteine. Conclusion: The results suggested that autophagy not only played an inhibitory role in the process of apoptosis but also could effectively attenuate lipid aggregation in THP-1 macrophages during HAC-SDT. As important intracellular mediators, the ROS generated by HAC-SDT also played a crucial role in initiating apoptosis and autophagy.

Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis.

The conventional photosensitizers used in photodynamic therapy (PDT), such as haematoporphyrin (HP), have not yet reached satisfactory therapeutic effects on port-wine stains (PWSs), due largely to the long-term dark toxicity. Previously we have showed that hypericin exhibited potent photocytotoxic effects on Roman chicken cockscomb model of PWSs. However, the molecular mechanism of hypericin-mediated photocytotoxicity remains unclear. In this study, we employed human umbilical vein endothelial cells (HUVECs) to investigate the hypericin-photolytic mechanism. Our study showed that hypericin-PDT induced reactive oxygen species (ROS), resulting in cell killings and an activation of the inflammatory response. Importantly, we have also discovered that photoactivated hypericin induced apoptosis by activating the mitochondrial caspase pathway and inhibiting the activation of the vascular endothelial growth factor-A (VEGF-A)-mediated PI3K/Akt pathway. Notably, we found that hypericin exhibited a more potent photocytotoxic effect than HP, and largely addressed the inconvenience issue associated with the use of HP. Thereby, hypericin may be a better alternative to HP in treating PWSs.

Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

GW25-e0820 Apoptosis of THP-1 macrophage-derived foam cells induced by 5-aminolevulinic acid-mediated sonodynamic therapy is mitochondria-caspase pathway predominant despite the participation of endoplasmic reticulum stress

In advanced atherosclerosis, chronic endoplasmic reticulum (ER) stress induces foam cells apoptosis and generates inflammatory reactions. Sonodynamic therapy (SDT) is a non-thermal synergistic method for cancer treatment utilizing low-intensity ultrasound and sonosensitizers. 5-Aminolaevulinic acid

Tirapazamine Sensitizes Hepatocellular Carcinoma Cells to Topoisomerase I Inhibitors via Cooperative Modulation of Hypoxia-Inducible Factor-1α

Topoisomerase I inhibitors are a class of anticancer drugs with a broad spectrum of clinical activity. However, they have limited efficacy in hepatocellular cancer. Here, we present in vitro and in vivo evidence that the extremely high level of hypoxia-inducible factor-1α (HIF-1α) in hepatocellular carcinoma is intimately correlated with resistance to topoisomerase I inhibitors. In a previous study conducted by our group, we found that tirapazamine could downregulate HIF-1α expression by decreasing HIF-1α protein synthesis. Therefore, we hypothesized that combining tirapazamine with topoisomerase I inhibitors may overcome the chemoresistance. In this study, we investigated that in combination with tirapazamine, topoisomerase I inhibitors exhibited synergistic cytotoxicity and induced significant apoptosis in several hepatocellular carcinoma cell lines. The enhanced apoptosis induced by tirapazamine plus SN-38 (the active metabolite of irinotecan) was accompanied by increased mitochondrial depolarization and caspase pathway activation. The combination treatment dramatically inhibited the accumulation of HIF-1α protein, decreased the HIF-1α transcriptional activation, and impaired the phosphorylation of proteins involved in the homologous recombination repair pathway, ultimately resulting in the synergism of these two drugs. Moreover, the increased anticancer efficacy of tirapazamine combined with irinotecan was further validated in a human liver cancer Bel-7402 xenograft mouse model. Taken together, our data show for the first time that HIF-1α is strongly correlated with resistance to topoisomerase I inhibitors in hepatocellular carcinoma. These results suggest that HIF-1α is a promising target and provide a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and tirapazamine in hepatocellular cancers.

Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion.

Decreased zinc levels in the macula are reported in patients with age-related macular degeneration, and the zinc chelator N,N,N',N'-tetrakis (2- pyridylmethyl) ethylenediamine) (TPEN) causes death of human retinal pigment epithelial (RPE) cells. The purpose of the present study was to investigate signal transduction pathways during cell death initiated by TPEN, using monkey RPE cells. RPE cells were cultured with TPEN. Activation of calpains and caspases, and proteolysis of their substrates were detected by immunoblotting. Incubation of calpain inhibitor SNJ-1945 or caspase inhibitor z-VAD-fmk was used to confirm activation of specific proteases. TPEN caused a time-dependent decrease in viable RPE cells. Cell death was accompanied by activation of calpain-1, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation and slightly inhibited caspase-9 activation. z-VAD-fmk inhibited caspases and calpain-1 activation. TPEN did not activate caspase-12. Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.

PPARγ mediates the effects of WIN55,212-2, an synthetic cannabinoid, on the proliferation and apoptosis of the BEL-7402 hepatocarcinoma cells

Cannabis sativa has long been used as a traditional medicine in China. Among its effective compounds are cannabinoids. This study determined the effect of WIN55,212-2 (WIN), a synthetic cannabinoid, on the BEL-7402 human hepatocellular carcinoma (HCC) cell line. The results showed that WIN could decrease the proliferation of BEL-7402 cells. Moreover, WIN could cause apoptosis of the cells via up-regulation of Bax expression, down-regulation of Bcl-2 expression, induction of the mitochondrial membrane potential, increase of caspase-3, -8 and -9 activities, and induction of the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). The WIN-induced apoptosis was accompanied by the up-regulation of PPARγ expression, the activation of PPARγ DNA binding activity, and a down-regulation of PPARγ target oncogene c-myc. Conversely, the effects of WIN could be attenuated by PPARγ antagonist GW9662, and the WIN induced PPARγ expression was partially attenuated by AM630, a cannabinoid receptor-2 antagonist, whereas the WIN-induced reduction of c-myc expression was partially restored by GW9662. Collectively, our results suggest that WIN can decrease the proliferation and cause apoptosis of the BEL-7402 cells via a mitochondrial-caspase pathway and mediated by PPARγ. These results may provide a basis for the application of WIN in HCC treatment.

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verified that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms.

Carbon monoxide inhibits apoptosis during cold storage and protects kidney grafts donated after cardiac death

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

Berberine, a Natural Product, Combined with Cisplatin Enhanced Apoptosis through a Mitochondria/Caspase-Mediated Pathway in HeLa Cells

Berberine, a main component of Coptidis Rhizoma, has been extensively studied and is known to exhibit multiple pharmacologic activities. In this study, we investigated whether the combination of berberine and cisplatin exhibited significant cytotoxicity in HeLa cells. Apoptosis was evaluated based on DNA fragmentation and cytofluorometrically with the annexin-V/propidium iodide labeling method. Combined treatment with berberine and cisplatin acted in concert to induce loss of mitochondrial membrane potential (Delta Psi m), release of cytochrome-c from mitochondria, and decreased expression of antiapoptotic Bcl-2, Bcl-x/L, resulting in activation of caspases and apoptosis. Further study showed that cell death induced by the combined treatment was associated with increased reactive oxygen species generation and lipid peroxidation. Moreover, we discovered that the combined treatment-induced apoptosis was mediated by the activation of the caspase cascade. These results indicated that the potential of cytotoxicity mediated through the mitochondria-caspase pathway is primarily involved in the combined treatment-induced apoptosis.

Cryoablation Induces Necrosis and Apoptosis in Lung Adenocarcinoma in Mice

This study evaluated cryoablation on subcutaneously transplanted tumors of lung adenocarcinoma LA795 in T739 mice in vivo, in an effort to assess the feasibility of cryoablation in treatment of NSCLC. Subcutaneously transplanted lung adenocarcinoma LA795 was implanted into T739 mice yielding tumors of approximately 2.5 cm in diameter. Following cryoablation, the various modes of cell death were studied: necrosis in the central frozen zone by light microscopy and apoptosis in periphery of the frozen zone by in situ end labeling (TUNEL). Bc1-2 and bax expression were detected by immunohistochemical SABC procedures, and the cleavage and activation of Caspase 3 and PARP in peripheral zone by Western blot. We find that in central cryoablated zone, necrosis was the dominant mode of cell death occurring at three hours and four days post-thaw. The first three-hour necrosis peak involved approximately 47% of the tumor while the four-day peak increased in volume to 68% of the tumor. In peripheral cryoablation zone, definite cell apoptosis could be observed by morphological examination under light microscope and TUNEL staining, peaking at 8-16 h after cryoablation. Immunohistochemical results yielded little change in bcl-2 protein expression before and after cryoablation. However, bax protein expression was up-regulated significantly after cryoablation. In addition, cleavage and activation of Caspase-3 and PARP occurred in the peripheral freeze zone after the treatment. It indicated that Cryoablation efficiently induces cell death both by necrosis and apoptosis. Cryoablation appears to induce apoptosis in the peripheral freeze zone through the intrinsic mitochondrial caspase pathway based on bax upregulation. This observation allows us to suggest that cryoablation may be combined with chemotherapy to increase cancer destruction.