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Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT‐29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT‐29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase‐dependent but also increases EndoG and AIF nuclear translocation in a caspase‐independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2‐mediated DNA damage‐responsive, caspase‐dependent/independent apoptotic effects. We uncovered a novel tumor‐inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC. [ABSTRACT FROM AUTHOR]

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This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg—94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg—94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of Hericium erinaceus (HE) on auditory function. Twenty-four 9-month-old male senescence-accelerated prone 8 (SAMP8) mice were randomly and equally assigned to the control group (group A) and two groups supplemented with 215.25 (group B) or 430.5 mg/kg BW of HE (group C) for 12 weeks. Compared with group A, group C had significantly smaller threshold shifts by auditory brainstem responses at the end of study. HE increased nerve growth factor (NGF) expressions in the spiral ganglion neurons (SGNs) and the temporal lobes, and also prevented outer hair cells (OHCs) loss in the middle turn of cochlea. In addition, group C had significantly lower apoptosis-inducing factor and poly (ADP-ribose) polymerase-1 expressions in the middle turn of cochlea, brainstem and temporal lobes. In conclusions, HE could prevent hearing degeneration possibly by increasing neurotrophic expression, preventing OHCs loss, and reducing the caspase-independent apoptosis signalings.

Breast cancer accounts for 1.4 million new cases every year. Triple-negative breast cancer (TNBC) is one the leading cause of mortality in developing countries and is associated with early age onset (under 40 years old). Chemotherapy has a poor success rate in patients with TNBC as compared to other types of breast cancers. It is due to the lack of expression of three validated molecular markers for breast cancer, the estrogen and progesterone receptors, and the amplification of HER-2/Neu. Therefore, a clear need exists for a greater understanding of TNBC at all levels and for the development of better therapies. We have studied the anti-tumor effects of a potential drug, maslinic acid, which can be extracted from olive oil industry waste. This natural product showed inhibitory effect at concentrations ranging from 30 to 50 µM within 24 h. It exhibited divergent effects in cell cycle progression for the MCF7 (estrogen positive) cell line when compared with TNBCs like MDA-MB-231 and MDA-MB-468. Also, maslinic acid treatment altered the mitochondrial membrane electrochemical potential and the reactive oxygen species (ROS) levels to cause a caspase-independent programmed cell death. In silico approaches and immunoblotting suggested the involvement of the MAPK pathway explaining the variability in cell cycle progression along with the apoptotic cell death caused by maslinic acid. [ABSTRACT FROM AUTHOR]

Acacetin, a bioflavanoid, contains anti-inflammatory and anti-cancer activities as shown in different experimental models. However, its anticancer potential and mechanism of action against colorectal cancer cells is largely unknown. Here, we have investigated the efficacy of acacetin using two colorectal adenocarcinoma SW480 and HCT-116 cell lines. Cell survival was examined by Trypan-blue exclusion and MTT assays, cell cycle analysis by FACS, apoptosis was assessed using Annexin V FITC assay and nuclear condensation by Hoechst staining, ROS level by DCFDA and Mitosox, and protein expression level by Western blotting. Acacetin reduced the cell survival and proliferation of both types of cells, and induced S- and G2-M phase arrest and also reduced the levels of β-catenin and its downstream target c-myc. Further, acacetin induced apoptosis as examined by Annexin-V FITC and nuclear condensation. It increased intracellular ROS production, especially mitochondrial ROS. Acacetin increased mitochondrial membrane potential depolarization and Bax:Bcl-2 ratio. Although significant changes in caspases -8 and -9 and PARP level was not observed, acacetin could induce the truncation and subsequent translocation of activated AIF from mitochondria to cytosol, which could further induce chromosomal breakage leading to apoptosis. In conclusion, Acacetin induces mitochondrial ROS-mediated cell death in a caspase-independent manner in SW480 and HCT-116 colon carcinoma cells by inducing apoptosis inducing factor (AIF), which may potentiate its anticancer and chemotherapeutic prospects against colorectal carcinoma. [ABSTRACT FROM AUTHOR]

Platinum-based drugs remain the reference treatment for gastric cancer (GC). However, the frequency of resistance, due to mutations in TP53 or alterations in the energy and redox metabolisms, impairs the efficacy of current treatments, highlighting the need for alternative therapeutic options. Here, we show that a cycloruthenated compound targeting the redox metabolism, RDC11, induces higher cytotoxicity than oxaliplatin in GC cells and is more potent in reducing tumor growth in vivo. Detailed investigations into the mode of action of RDC11 indicated that it targets the glutathione (GSH) metabolism, which is an important drug resistance mechanism. We demonstrate that cycloruthenated complexes regulate the expression of enzymes of the transsulfuration pathway via the Unfolded Protein Response (UPR) and its effector ATF4. Furthermore, RDC11 induces the expression of SLC7A11 encoding for the cystine/glutamate antiporter xCT. These effects lead to a lower cellular GSH content and elevated oxygen reactive species production, causing the activation of a caspase-independent apoptosis. Altogether, this study provides the first evidence that cycloruthenated complexes target the GSH metabolism, neutralizing thereby a major resistance mechanism towards platinum-based chemotherapies and anticancer immune response. • A Cycloruthenated compound induces higher cytotoxicity than oxaliplatin in gastric cancer cells and strongly reduces tumor growth in vivo. • Anti-cancer cycloruthenated compounds target the redox metabolism in gastric cancer cells. • Cycloruthenated compounds target the glutathione (GSH) metabolism through the Unfolded Protein Response (UPR), and its effector ATF4. • These effects lead to a lower cellular GSH content and higher oxygen reactive species production, leading to caspase-independent apoptosis. [ABSTRACT FROM AUTHOR]

Justicidin A (JA), a natural lignan, was isolated from the whole plant of Justicia procumbens, one of the most popular traditional Chinese medicines in China and Taiwan. Gene expression signatures of JA from human colorectal cancer HT-29 and hepatocellular carcinoma Hep 3B cells were used to query connectivity map. A NF-κB suppressor (15-delta prostaglandin J2) was predicted to display similar molecular action of JA. In JA-treated HT-29 cells, suppression of nuclear NF-κB expression and decrease of NF-κB DNA-binding activity were indeed observed. The classical pathway was involved in JA-induced inhibition of NF-κB, characterized by decreases in phosphorylation of AKT, IκB kinase (IKK)-β/IKK-α, and IκB-α. Furthermore, JA caused significant translocation of apoptosis-inducing factor and endonuclease G, caspase-independent apoptotic signaling molecules, from the mitochondrial to the nuclei. Our data suggest anti-inflammatory and cytotoxic mechanisms of JA, and using gene expression signatures to identify novel molecular actions of phytochemicals is an effective approach.

The involvement of glutamate in neuronal cell death in neurodegenerative diseases and neurotrauma is mediated through excitotoxicity or oxytosis. The latter process induces oxidative stress via glutamate-mediated inhibition of cysteine transporter xCT, leading to depletion of the cellular glutathione pool. Mitochondrial damage, loss of mitochondrial membrane potential (MMP), and depletion of energy metabolites have been shown in this process. The Voltage-Dependent Anion Channel-1 (VDAC1) is one of the main components of the mitochondrial outer membrane and plays a gatekeeping role in mitochondria-cytoplasm transport of metabolites. In this study, we explored the possible participation of VDAC-1 in the pathophysiology of oxytosis. Administration of glutamate in HT22 cells that lack the glutamate ionotropic receptors induced an upregulation and oligomerization of VDAC1. This was associated with an increase in ROS and loss of cell survival. Glutamate-mediated oxytosis in this model also decreased MMP and promoted ATP depletion, resulting in translocation of cytochrome c (cyt C) and apoptosis inducing factor (AIF) from mitochondria into the cytosol. This was also accompanied by cleavage of AIF to form truncated AIF. Inhibition of VDAC1 oligomerization using 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), significantly improved the cell survival, decreased the ROS levels, improved mitochondrial functions, and decreased the mitochondrial damage. Notably, DIDS also inhibited the mitochondrial fragmentation caused by glutamate, indicating the active role of VDAC1 oligomerization in the process of mitochondrial fragmentation in oxytosis. These results suggest a critical role for VDAC1 in mitochondrial fragmentation and its potential therapeutic value against glutamate-mediated oxidative neurotoxicity. [ABSTRACT FROM AUTHOR]

Antitumour effects of α-tomatine were studied during 24 hours in a panel of human colon cancer cells. α-Tomatine proved to induce dose-dependent cytotoxicity resulting in morphologically varying cell demise with predominantly occurring necrotic phenotypes and minor presence of cells dying via caspase-independent apoptosis. Analyses into the nature of mechanisms responsible for activation of cell death revealed that following α-tomatine treatment, changes in RIP3 and RIP1 protein expression take place along with cyclophilin D mitochondrial accumulation. Moreover, in treated cells, lysosomal membrane permeabilization as well as mitochondrial perturbation with subsequent mitochondrial release of apoptosis-inducing factor (AIF) contributes to the execution of diverse death phenotypes possibly via enhanced activity JNK but in the absence of significant oxidative stress. Thus α-tomatine exhibits significant antitumour activity in colon cancer cells via targeting their membranous compartments and inducing both necroptosis and apoptosis.

Background/Aims: Phosphatidylinositol (PI) regulates a variety of cell processes. The present study investigated the antitumor action of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DPPI) on human malignant pleural mesothelioma (MPM) cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells. Methods: MTT assay, TUNEL staining, flow cytometry using propidium iodide (PI) and annexin V (AV), enzymatic caspase assay, and nuclear staining using DAPI were carried out, and mitochondrial membrane potentials and intracellular distribution of apoptosis-inducing factor (AIF) were monitored in cells with and without the siRNA silencing the Bid-targeted gene. Results: Both DOPI and DPPI reduced cell viability for all the investigated MPM cell lines in a concentration (0.01-100 µM)-dependent manner. DOPI and DPPI significantly increased TUNEL-positive cells and the population of PI-negative/AV-positive and PI-positive/AV-positive cells, corresponding to early apoptosis and late apoptosis/secondary necrosis, respectively. DOPI and DPPI perturbed mitochondrial membrane potentials in MSTO-211H cells, but no significant activation of caspase-3, -4, -8, and -9 was obtained. DOPI and DPPI upregulated expression of Bid in MSTO-211H cells. DOPI and DPPI significantly increased nuclear localization of AIF without affecting expression of the mRNAs and proteins in MSTO-211H cells, which was inhibited by knocking-down Bid. In the DAPI staining, nuclear fragmentation and condensation were found. Conclusion: The results of the present study indicate that DOPI and DPPI facilitate Bid-mediated AIF release from the mitochondria, to accumulate AIF in the nucleus and induce caspase-independent apoptosis of MPM cells.

Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca2+-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca2+ levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca2+ accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca2+ overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and in vivo tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish. [ABSTRACT FROM AUTHOR]

For patients with refractory or relapsed Burkitt lymphoma (BL), no standard therapy is available for second-line treatment to date. Nonfunctional caspases-dependent apoptosis pathways, inactivating p53 mutations and pro-survival autophagy prevent activity of conventional chemotherapy. Thus, new drugs bypassing these mechanisms of resistance are required. Here, we investigated the efficacy of the marine natural compound frondoside A (FrA) in eight BL cell lines. FrA revealed cytotoxic effects in all cell lines tested including the multiresistant CA46 cells. Remarkably, FrA induced caspases- and p53-independent apoptosis, which was characterized by decreased expression of antiapoptotic survivin and Bcl-2, mitochondria targeting (release of cytochrome C, HtrA2/Omi and the apoptosis-inducing factor (AIF), and altered production of ROS) and translocation of AIF to the nuclei. In addition, signs of inhibition of pro-survival autophagy were observed. Thus, FrA is a promising candidate for the treatment of refractory or relapsed BL revealing resistances to standard therapies. [ABSTRACT FROM AUTHOR]

The aim of this study is to determine the behavior of relative expression of Bcl-2, caspase-8, caspase-9, and caspase-3 genes of/in SK-MEL-3 cancer cells and explore molecular mechanisms responsible for the apoptosis response during an in vitro photodynamic therapy (PDT) with Zinc Phthalocyanine (ZnPc) using different doses of the light source. In this study, firstly the cytotoxic effects of ZnPc-PDT on SK-MEL-3 cells were evaluated. By irradiating the laser, ZnPc induced a significant amount of apoptosis on SK-MEL-3 cells in three IC 50 s including 0.064 ± 0.01, 0.043 ± 0.01, and 0.036 ± 0.01 μg/mL at the doses of 8, 16, and 24 J/cm 2 , respectively. Moreover, flow cytometry and QRT-PCR experiments were done. The high percentage of apoptotic cells was seen in the early apoptosis stage. The expression of Bcl-2 and caspase-8 genes at all doses of laser experienced an obvious reduction in comparison to the control group. On the other hand, although the expression of caspase-9 and caspase-3 genes remains almost constant at 8 J/cm 2 , but they faced an increment at 16 and 24 J/cm 2 doses. These data reveal caspase-dependent apoptosis in high and caspase-independent apoptosis in low doses of laser. Based on the results of present work, it can be suggested that the dose of the light source is a key factor in induction of caspase-dependent and caspase-independent apoptosis pathways following PDT. [ABSTRACT FROM AUTHOR]

Purpose Fucoidan, a complex, sulfated polysaccharide obtained from brown seaweed, exerted anticancer activity through the down-regulation of β-catenin signaling in mouse breast cancer cells in our previous study. This study examines the anti‐cancer effects of fucoidan as well as its underlying molecular mechanisms in the human triple negative breast cancer (TNBC) cell line and in 7,12-dimethylbenz[a]anthracene (DMBA)-induced experimental mammary carcinogenesis in rats. Methods in vitro studies, fluorescent staining, flow cytometry and Western blotting were performed to analyze apoptosis and protein expression in human breast cancer MDA-MB-231 cells. In vivo intervention experiments were conducted with Sprague Dawley (SD) rats with DMBA-induced breast cancer. Tumor volumes and weights were measured. Results in vitro fucoidan treatment inhibited proliferation and induced apoptosis in MDA-MB-231 cells. Western blotting detected that Cyt C and Smac were released into the cell cytoplasm and that caspase-3 and caspase-9 were activated in MDA-MB-231 cells. The levels of AIF and EndoG were significantly increased in the cytoplasm and in the nuclei by fucoidan. These data show that fucoidan induced caspase-dependent and caspase-independent apoptosis. Moreover, fucoidan treatment down-regulated the expression of Bid, Bcl-2 and Bcl-xl and up-regulated the level of Bax. In vivo, fucoidan supplementation decreased the mean tumor weight. Discussion: Results from the in vivo and in vitro experiments both showed that fucoidan decreased the levels of p-PI3K, p-AKT and p-GSK-3β (Ser9) in breast cancer. The level of β-catenin was also decreased. These results suggest that fucoidan can inhibit MDA-MB-231 human breast cancer cells and DMBA-induced tumors in rats by down-regulating the PI3 K/AKT/GSK3β pathway. This study provides experimental evidence that elucidates the mechanism of antitumor effect of fucoidan and clarifies the mechanism of the effect of fucoidan on the regulation of β-catenin. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by relatively selective death of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). 6-Hydroxydopamine (6-OHDA), a neurotoxin that causes the death of DA neurons, is commonly used to produce experimental PD model in rodents. Accumulating evidences suggest that caspase-independent apoptotic programmed cell death (PCD) could also be involved in the progression of various neurodegenerative diseases in addition to caspase-dependent neuronal PCD. Apoptosis-inducing factor (AIF), a mitochondrial intermembrane oxidoreductase, has been identified as a key protein implicated in caspase-independent apoptosis. However, little is known about the role of AIF in death of nigral DA neurons in PD. Therefore, we undertook this study in an effort to clarify the involvement of AIF in DA neuronal death by 6-OHDA administration. Ten and twenty micrograms of 6-OHDA was infused into the medial forebrain bundle (MFB) unilaterally, and the experimental rats were sacrificed at various time point. The DA neuronal loss was identified in the ipsilateral SN in the dose-dependent manner by using NeuN and tyrosine hydroxylase immunohistochemical staining and western blot assay. Numerous degenerating neurons, showing apoptotic features which are characterized by the shrunken nuclei with eosinophilic perikarya were observed in the ipsilateral SNpc. Activating transcription factor 3 (ATF3), the specific marker for neuronal damage, was expressed in the ipsilateral DA neurons only. Immunohistochemistry and immunofluorescence staining demonstrated that nuclear localization of AIF in the ipsilateral degenerating DA neurons. These results suggest that AIF could induce DA neuronal death by caspase-independent apoptosis in 6-OHDA treated model, although other cell death cascades should not be rule out. [ABSTRACT FROM AUTHOR]

Justicidin A (JA), a natural lignan, was isolated from the whole plant of Justicia procumbens , one of the most popular traditional Chinese medicines in China and Taiwan. Gene expression signatures of JA from human colorectal cancer HT-29 and hepatocellular carcinoma Hep 3B cells were used to query connectivity map. A NF-κB suppressor (15-delta prostaglandin J2) was predicted to display similar molecular action of JA. In JA-treated HT-29 cells, suppression of nuclear NF-κB expression and decrease of NF-κB DNA-binding activity were indeed observed. The classical pathway was involved in JA-induced inhibition of NF-κB, characterized by decreases in phosphorylation of AKT, IκB kinase (IKK)-β/IKK-α, and IκB-α. Furthermore, JA caused significant translocation of apoptosis-inducing factor and endonuclease G, caspase-independent apoptotic signaling molecules, from the mitochondrial to the nuclei. Our data suggest anti-inflammatory and cytotoxic mechanisms of JA, and using gene expression signatures to identify novel molecular actions of phytochemicals is an effective approach. [ABSTRACT FROM AUTHOR]

The toxicity of melamine has attracted much attention since the recent outbreaks of renal injury in pets and infants. Previous studies indicated that melamine by itself had low toxicity, whereas a mixture of melamine and cyanuric acid (M+CA) could cause serious renal damage. At present, most researches on the toxicity of M+CA are focused on the kidney. However, little is known about the adverse effects of this mixture on the reproductive system. In the present study, the toxicity of M+CA to testes was investigated. Immature male mice were orally dosed with 0, 0.6, 3, and 15 mg kg−1 d−1 of a 1:1 M+CA for 28 d. Pathological changes occurred in germ cells, such as loose arrangement, reduced numbers and karyopyknosis, indicating that this mixture was toxic to spermatogenesis. Compared with the control group, the TUNEL-positive germ cells increased significantly and the ratio of Bcl-2 to Bax, total antioxidant capacity and superoxide dismutase activity decreased significantly in the 3 and 15 mg kg−1 d−1 M+CA treated group, while the activities of caspase-3, caspase-8 and caspase-9 remained unchanged. The results suggest that M+CA can induce apoptosis in the mice testes. The downregulation of Bcl-2/Bax and oxidative stress may play a pivotal role in the induction of apoptosis by M+CA in mice testes.

Colorectal cancer (CRC) is recognized as the third most common malignancy and the second most deadly in highly developed countries. Although the treatment of CRC has improved in the past decade, the mortality rate of CRC is still increasing. Amentoflavone, one of the flavonoids detected in medical plants, is reported to possess potential anticancer properties in various cancers. However, its role in CRC has not been studied. This study aimed to investigate the role and underlying mechanism of amentoflavone on CRC in vitro and in vivo. We identified the cytotoxicity, apoptosis effect, cell cycle alteration, DNA damage induction and tumor progression inhibition of amentoflavone in HT-29 model by using MTT assay, flow cytometry, immunofluorescence (IF) staining, Western blotting and animal experiments. Amentoflavone induced cytotoxicity is caused by triggering G1 arrest, DNA damage and apoptosis in HT-29 cells. The expression of cyclin D1, CDK4 and CDK6 was decreased by amentoflavone; in contrast, the phosphorylation of ATM and CHK2 and the expression of p21 and p27 were increased. The apoptosis induction of amentoflavone in CRC is not only caspase-dependent but also increases EndoG and AIF nuclear translocation in a caspase-independent manner. Importantly, the apoptosis induction of amentoflavone is not affected by the activity of p53 in CRC. Amentoflavone suppressed the progression of CRC by initiating G1 arrest and ATM/CHK2-mediated DNA damage-responsive, caspase-dependent/independent apoptotic effects. We uncovered a novel tumor-inhibitory role of amentoflavone in CRC that is not associated with p53 activity, which may serve as a potential treatment for CRC., (© 2023 Wiley Periodicals LLC.)

Antitumour effects of α-tomatine were studied during 24 hours in a panel of human colon cancer cells. α-Tomatine proved to induce dose-dependent cytotoxicity resulting in morphologically varying cell demise with predominantly occurring necrotic phenotypes and minor presence of cells dying via caspase-independent apoptosis. Analyses into the nature of mechanisms responsible for activation of cell death revealed that following α-tomatine treatment, changes in RIP3 and RIP1 protein expression take place along with cyclophilin D mitochondrial accumulation. Moreover, in treated cells, lysosomal membrane permeabilization as well as mitochondrial perturbation with subsequent mitochondrial release of apoptosis-inducing factor (AIF) contributes to the execution of diverse death phenotypes possibly via enhanced activity JNK but in the absence of significant oxidative stress. Thus α-tomatine exhibits significant antitumour activity in colon cancer cells via targeting their membranous compartments and inducing both necroptosis and apoptosis. [ABSTRACT FROM AUTHOR]

Background/Aims: Phosphatidylinositol (PI) regulates a variety of cell processes. The present study investigated the antitumor action of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myoinositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DPPI) on human malignant pleural mesothelioma (MPM) cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells. Methods: MTT assay, TUNEL staining, flow cytometry using propidium iodide (PI) and annexin V (AV), enzymatic caspase assay, and nuclear staining using DAPI were carried out, and mitochondrial membrane potentials and intracellular distribution of apoptosisinducing factor (AIF) were monitored in cells with and without the siRNA silencing the Bidtargeted gene. Results: Both DOPI and DPPI reduced cell viability for all the investigated MPM cell lines in a concentration (0.01-100 |jM)-dependent manner. DOPI and DPPI significantly increased TUNEL-positive cells and the population of PI-negative/AV-positive and PI-positive/ AV-positive cells, corresponding to early apoptosis and late apoptosis/secondary necrosis, respectively. DOPI and DPPI perturbed mitochondrial membrane potentials in MSTO-211H cells, but no significant activation of caspase-3, -4, -8, and -9 was obtained. DOPI and DPPI upregulated expression of Bid in MSTO-211H cells. DOPI and DPPI significantly increased nuclear localization of AIF without affecting expression of the mRNAs and proteins in MSTO-211H cells, which was inhibited by knocking-down Bid. In the DAPI staining, nuclear fragmentation and condensation were found. Conclusion: The results of the present study indicate that DOPI and DPPI facilitate Bid-mediated AIF release from the mitochondria, to accumulate AIF in the nucleus and induce caspase-independent apoptosis of MPM cells. [ABSTRACT FROM AUTHOR]

Apoptotic signaling plays an important role in the development and maintenance of healthy skeletal muscle. However, dysregulation of apoptotic signals in skeletal muscle is associated with atrophy and loss of function. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is highly expressed in skeletal muscle; however, its role in this tissue has yet to be elucidated. To investigate whether ARC deficiency has morphological, functional, and apoptotic consequences, skeletal muscle from 18 week-old wild-type and ARC knockout (KO) mice was studied. In red muscle (soleus), we found lower maximum tetanic force, as well as a shift towards a greater proportion of type II fibers in ARC KO mice. Furthermore, the soleus of ARC KO mice exhibited lower total, as well as fiber type-specific cross sectional area in type I and IIA fibers. Interestingly, these changes in ARC KO mice corresponded with increased DNA fragmentation, albeit independent of caspase or calpain activation. However, cytosolic fractions of red muscle from ARC KO mice had higher apoptosis inducing factor content, suggesting increased mitochondrial-mediated, caspase-independent apoptotic signaling. This was confirmed in isolated mitochondrial preparations, as mitochondria from skeletal muscle of ARC KO mice were more susceptible to calcium stress. Interestingly, white muscle from ARC KO mice showed no signs of altered apoptotic signaling or detrimental morphological differences. Results from this study suggest that even under basal conditions ARC influences muscle apoptotic signaling, phenotype, and function, particularly in slow and/or oxidative muscle. [ABSTRACT FROM AUTHOR]

Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Nonthermal atmospheric pressure plasma can be applied to living cells and tissues and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only directly, but also by indirect treatment with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the inhibitory effects of PAM on A549 cell survival and elucidate the signaling mechanisms responsible for cell death. PAM maintained its ability to suppress cell viability for at least 1 week when stored at −80 °C. The severity of PAM-triggered cell injury depended on the kind of culture medium used to prepare the PAM, especially that with or without pyruvate. Hydrogen peroxide (H 2 O 2 ) and/or its derived or cooperating reactive oxygen species reduced the mitochondrial membrane potential, downregulated the expression of the antiapoptotic protein Bcl 2 , activated poly(ADP-ribose) polymerase-1, and released apoptosis-inducing factor from mitochondria with endoplasmic reticulum stress. However, the activation of caspase 3/7 and attenuation of cell viability by the addition of caspase inhibitor were not observed. The accumulation of adenine 5′-diphosphoribose as a product of the above reactions activated transient receptor potential melastatin 2, which elevated intracellular Ca 2+ levels and subsequently led to cell death. These results demonstrated that H 2 O 2 and/or other reactive species in PAM disturbed the mitochondrial–nuclear network in cancer cells through a caspase-independent apoptotic pathway. Moreover, damage to the plasma membrane by H 2 O 2 -cooperating charged species not only induced apoptosis, but also increased its permeability to extracellular reactive species. These phenomena were also detected in PAM-treated HepG2 and MCF-7 cells. [ABSTRACT FROM AUTHOR]

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: The newly synthesized phosphatidylinositol (PI) derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidyl-D-1-inositol (diDCP-LA-PI) significantly enhanced protein phosphatase 2A (PP2A) activity in the cell-free assay. This prompted to assess the antitumor effect of diDCP-LA-PI. diDCP-LA-PI attenuated phosphorylation of mitogen-activated protein kinase (MAPK) kinase (MEK) in Lu65 human lung cancer and MKN28 human gastric cancer cells with high MEK activity. diDCP-LA-PI reduced cell viability in Lu65 and MKN28 cells, but otherwise such effect was not found in 786-O human renal cancer and HUH-7 human hepatoma cells with relatively low MEK activity. For Lu65 and MKN28 cells diDCP-LA-PI increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells, but no significant activation of caspase-3, -8, or -9 was obtained. For MKN28 cells diDCP-LA-PI-induced reduction of MEK phosphorylation and cell viability was prevented by knocking-down PP2Ac. Taken together, these results indicate that diDCP-LA-PI induces caspase-independent apoptosis of Lu65 and MKN28 human cancer cells, for the latter cells by suppressing MEK activity through PP2A-catalyzed dephosphorylation. [Copyright &y& Elsevier]

Highlights: [•] We synthesized the acetyl derivative of the antitumor agent apicularen A. [•] Apicularen A acetate induces cytotoxicity and sub-G1 accumulation in HM7 cells. [•] Apicularen A acetate triggers caspase-independent cell death via AIF translocation. [•] Apicularen A acetate disrupts the microtubule networks by tubulin down-regulation. [•] Substitution of functional groups of drugs may be a good strategy for cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract: Inactivation of epidermal growth factor receptor (EGFR) family members are prime targets for cancer therapy. Here, we show that tephrosin, a natural rotenoid which has potent antitumor activities, induced internalization of EGFR and ErbB2, and thereby induced degradation of the receptors. Treatment of HT-29 cells with tephrosin inhibited both the ligand-induced and constitutive phosphorylation of EGFR, ErbB2 and ErbB3, and concomitantly suppressed the activation of the downstream signaling molecules such as Akt and Erk1/2 mitogen-activated protein kinase (MAPK). Tephrosin caused internalization of EGFR and ErbB2 into vehicles, which resulted in degradation of the receptors. This degradation was blocked by the lysosomal inhibitor, chloroquine. We also showed that tephrosin induced apoptosis. Tephrosin did not induce the proteolytic processing of caspase-3 and poly(ADP-ribose) polymerase (PARP), but did nuclear translocation of apoptosis-inducing factor (AIF), suggesting that tephrosin may induce caspase-independent apoptosis. These findings provide the first evidence that tephrosin could exert antitumor effects by inducing internalization and degradation of inactivated EGFR and ErbB2 in human colon cancer cells. [Copyright &y& Elsevier]

The article presents a study which evaluates whether the activation of an alternative, caspase-independent cell death pathway could promote death in chemoresistant ovarian cancer cells. Result shows that NV-128, an Isoflavone derivative inhibits mammalian target of rapamycin (mTOR) and promotes caspase-independent cell death. They concluded that the use of NV-128 may have beneficial effects in patients with chemoresistant ovarian cancer.

RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.Cancer Gene Therapy (2009) 16, 13–19; doi:10.1038/cgt.2008.54; published online 25 July 2008 [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome proliferator-activated receptor γ (PPARγ) agonists have been reported to induce apoptosis in a variety of cell types including renal proximal epithelial cells. However, the underlying mechanism of cell death induced by PPARγ agonists has not been clearly defined in renal proximal tubular cells. This study was therefore undertaken to determine the mechanism by which ciglitazone, a synthetic PPARγ agonist, induces apoptosis in opossum kidney (OK) cells, an established renal epithelial cell line. Ciglitazone treatment induced apoptotic cell death in a dose- and time-dependent manner. Ciglitazone caused a transient activation of ERK and sustained activation of p38 MAP kinase. Ciglitazone-mediated cell death was attenuated by the p38 inhibitor SB203580 and transfection of dominant-negative form of p38, but not by the MEK inhibitor U0126, indicating that p38 MAP kinase activation is involved in the ciglitazone-induced cell death. Although ciglitazone-induced caspase-3 activation, the ciglitazone-mediated cell death was not affected by the caspase-3 inhibitor DEVD-CHO. Ciglitazone-induced mitochondrial membrane depolarization and apoptosis-inducing factor (AIF) nuclear translocation and these effects were prevented by the p38 inhibitor. These results suggest that ciglitazone induces caspase-independent apoptosis through p38 MAP kinase-dependent AIF nuclear translocation in OK renal epithelial cells. [Copyright &y& Elsevier]

XAF1 is a newly identified tumor-suppressor gene that can antagonize XIAP and sensitize cells to other cell death triggers. In this study, we utilized ZD55, a conditionally replicative adenovirus (CRAd) similar to ONYX-015 as the vector to transfer XAF1 into the tumor cells to evaluate its antitumor efficacy in vitro and in vivo. Potent and specific cytopathic effect (CPE) was observed upon infection with ZD55-XAF1 in tumor cell lines. Importantly, ZD55-XAF1 exhibited a superior suppression of tumor growth in an animal model of colorectal carcinoma in nude mice compared with Ad-XAF1 (E1-deleted replication-defective viral) and ONYX-015. Complete eradication of the established tumors was observed in four of eight mice. Our data also showed that infection with ZD55-XAF1 resulted in caspase-independent apoptosis. Although caspase-3, poly(ADP-ribose) polymerase were mildly activated in response to ZD55-XAF1 infection, pretreatment with pan-caspase inhibitor hardly influence its apoptosis-inducing activity. In summary, our study strongly suggested that ZD55-XAF1 could serve as an effective gene-virotherapy strategy and has highly potential against human cancers.Cancer Gene Therapy (2007) 14, 82–90. doi:10.1038/sj.cgt.7700992; published online 29 September 2006 [ABSTRACT FROM AUTHOR]

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a naturally occurring cyclopentenone metabolite of prostaglandin D2 (PGD2) and is known as a specific potent ligand for the peroxisome proliferators activator receptor-γ (PPARγ). 15d-PGJ2 inhibits cell growth and induces apoptosis in a number of different cancer cells. However, the underlying mechanism by which 15d-PGJ2 induces cell death remains to be defined. The present study was undertaken to determine the effect of 15d-PGJ2 on cell death in A172 human glioma cells. 15d-PGJ2 caused reactive oxygen species (ROS) generation. 15d-PGJ2-induced ROS production and cell death were prevented by the antioxidant N-acetylcysteine. Activation of mitogen-activated protein kinases (MAPK) was not observed in cells treated with 15d-PGJ2 and inhibitors of MAPK subfamilies also were not effective in preventing 15d-PGJ2-induced cell death. 15d-PGJ2 treatment caused mitochondrial dysfunction, as evidenced by depolarization of mitochondrial membrane potential. 15d-PGJ2 induced caspase activation at 24 h of treatment, but the 15d-PGJ2-induced cell death was not prevented by caspase inhibitors. The antiapoptotic protein XIAP levels and release of apoptosis inducing factor (AIF) into the cytosol were not altered by 15d-PGJ2 treatment. Taken together, these findings indicate that 15d-PGJ2 triggers cell death through a caspase-independent mechanism and ROS production and disruption of mitochondrial membrane potential play an important role in the 15d-PGJ2-induced cell death in A172 human glioma cells. [ABSTRACT FROM AUTHOR]