Mitochondria-mediated Apoptotic Cell Death Research Articles

A secreted form of chorismate mutase (Rv1885c) in Mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages

BackgroundMycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown.MethodsA tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG.ResultsDeletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death.ConclusionsOur findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.

Tebuconazole Induces ER-Stress-Mediated Cell Death in Bovine Mammary Epithelial Cell Lines.

Tebuconazole (TEB) is a triazole fungicide used to increase crop production by controlling fungi, insects, and weeds. Despite their extensive use, people are concerned about the health risks associated with pesticides and fungicides. Numerous studies have defined the cellular toxicity of triazole groups in pesticides, but the mechanisms of TEB toxicity in bovine mammary gland epithelial cells (MAC-T cells) have not yet been studied. Damage to the mammary glands of dairy cows directly affects milk production. This study investigated the toxicological effects of TEB on MAC-T cells. We found that TEB decreases both cell viability and proliferation and activates apoptotic cell death via the upregulation of pro-apoptotic proteins, such as cleaved caspases 3 and 8 and BAX. TEB also induced endoplasmic reticulum (ER) stress via the upregulation of Bip/GRP78; PDI; ATF4; CHOP; and ERO1-Lα. We found that TEB induced mitochondria-mediated apoptotic MAC-T cell death by activating ER stress. This cell damage eventually led to a dramatic reduction in the expression levels of the milk-protein-synthesis-related genes LGB; LALA; CSN1S1; CSN1S2; and CSNK in MAC-T cells. Our data suggest that the exposure of dairy cows to TEB may negatively affect milk production by damaging the mammary glands.

Abstract 5800: Anti-metastatic potential of pitavastatin in triple-negative breast cancer via targeting breast cancer stem-like properties and STAT3 signaling

Abstract Triple-negative breast cancer (TNBC) is the most deadly and aggressive phenotype, with a higher rate of metastatic recurrence. TNBC does yet have a suitable treatment option other than cytotoxic anticancer drugs. Although pitavastatin has been shown to exert anti-proliferative effects and cytotoxicity in various types of cancer cells, the precise mechanisms underlying pitavastatin’s anti-cancer effects in TNBC have not been fully elucidated. We sought to investigate the mechanism of pitavastatin-induced apoptosis and its effects on cancer stem cell (CSC)-like characteristics in TNBC. Exposure to pitavastatin induced mitochondria-mediated apoptotic cell death in BT549 and 4T1 cells. Mitochondrial dysfunction was accompanied with a robust production of reactive oxygen species (ROS) and collapse of mitochondrial membrane potential (MMP), resulting in subsequent activation of caspase-3/-7 and PARP cleavage. Pitavastatin effectively suppressed CSC-like properties in TNBC via targeting CD44+/CD24- and CD49f+/CD24- phenotypes, as well as impediment of mammosphere formation in vitro. This phenomenon was accompanied with dysregulation of STAT3 survival pathway, concomitant with significant downregulation of cyclin D1, survivin and vimentin. Pitavastatin effectively targets both the proliferating TNBC tumor cells and CSCs via the dysregulation of STAT3 and suppression of CSC-like properties, markedly reducing angiogenesis and tumor growth, coinciding with decreased Ki-67 expression. It is noteworthy that pitavastatin considerably suppressed metastasis, coinciding with significant reduction of MMP-2, MMP-9 and VEGF in the circulating blood of mice. Our findings highlight that pitavastatin may be potentially effective for the treatment of metastatic TNBC. Citation Format: Dongmi Ko, Juyeon Seo, Seongjae Kim, Soeun Park, Minsu Park, Kee Dal Nam, Yong koo Kang, Sora Seock, Eunsun Jung, Yoon-Jae Kim, Jaeyoun Park, Ji Young Kim, Jae Hong Seo. Anti-metastatic potential of pitavastatin in triple-negative breast cancer via targeting breast cancer stem-like properties and STAT3 signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5800.

MDM2 inhibition is synthetic lethal with PTEN loss in colorectal cancer cells via the p53-dependent mechanism.

Colorectal cancer (CRC) driven by PTEN deficiency exhibits high risk of metastasis, advancement of tumor stages and chemotherapy resistance, where no effective therapy has been developed. In this study, we performed a synthetic lethal drug screening in CRC and found that PTEN-deficient CRC cells are highly vulnerable to MDM2 inhibition. MDM2 inhibitor treatment or its silencing selectively inhibited the growth of PTEN-deficient CRC in vitro and in mice models. Mechanistically, PTEN loss increased the level of active AKT and subsequently increased MDM2 phosphorylation, thereby limiting the p53 functions in PTEN-/- CRC cells. MDM2 inhibition in turn activated p53 in CRC, particularly in PTEN-/- CRC cells. The synthetic lethal effect of MDM2 inhibitor was largely dependent on p53, because p53 silenced cells or cells lacking p53 failed to exhibit synthetic lethality in PTEN-deficient cells. We further showed that MDM2 inhibition led to the p53-dependent reversal of Bcl2-Bax ratio, which contributed to mitochondria-mediated apoptotic cell death in PTEN-deficient CRC. This study suggests that pharmacological targeting of MDM2 could be a potential therapeutic strategy for PTEN-deficient CRC.

Modulation of calcium signaling by nanosecond electric pulses and cell death through apoptosis in A549 lung cancerous cells

Calcium ion (Ca2+), which serves as one of the important signaling agents, is imperative to control normal cell function. The altered Ca2+ signaling is known to play a crucial role in the resistance of cancerous cells against apoptosis. Herein, nanosecond pulsed electric field (nsPEF) was applied to non-small lung cancerous cells A549, and Ca2+ mobilization along with mitochondria-mediated apoptotic cell death that accompanies morphological changes was investigated. In the presence of nsPEF, intracellular mobilization of Ca2+ ions through the efflux from endoplasmic reticulum storage was dependent on the pulse-width of the applied field. The field-induced Ca2+ mobilization has shown to correlate with the superoxide anion (O2−) generation, which probably occurred in mitochondria utilizing the nicotinamide adenine dinucleotide (NADH), as supported by the field-induced change in autofluorescence intensity and lifetime of NADH. Mitochondrial dysfunction and O2− generation, which promoted apoptotic cell death, were also induced by nsPEF with strong correlation with intracellular Ca2+ ions.

NIR-II Emissive Ru(II) Metallacycle Assisting Fluorescence Imaging and Cancer Therapy.

Despite the success of emissive Ruthenium (Ru) agents in biomedicine, problems such as the visible-light excitation/emission and single chemo- or phototherapy modality still hamper their applications in deep-tissue imaging and efficient cancer therapy. Herein, an second nearinfrared window (NIR-II) emissive Ru(II) metallacycle (Ru1000, λem =1000nm) via coordination-driven self-assembly is reported, which holds remarkable deep-tissue imaging capability (≈6mm) and satisfactory chemo-phototherapeutic performance. In vitro results indicate Ru1000 displays promising cellular uptake, good cancer-cell selectivity, attractive anti-metastasis properties, and remarkable anticancer activity against various cancer cells, including cisplatin-resistant A549 cells (IC50 =3.4×10-6 m vs 92.8×10-6 m for cisplatin). The antitumor mechanism could be attributed to Ru1000-induced lysosomal membrane damage and mitochondrial-mediated apoptotic cell death. Furthermore, Ru1000 also allows the high-performance in vivo NIR-II fluorescence imaging-guided chemo-phototherapy against A549 tumors. This work may provide a paradigm for the development of long-wavelength emissive metallacycle-based agents for future biomedicine.

Quercus acutissima Carruth. root extract triggers apoptosis, autophagy and inhibits cell viability in breast cancer cells

Ethnopharmacological relevanceThe bark of Quercus acutissima Carruth. (QA) has long been used by Chinese people to treat noncancerous growths and cancerous ailments. It was traditionally used by Chinese folk to inhibit tumor proliferation in cancerous treatment, but the specific mechanism remain to be elucidated. Aim of the studyThis study investigated the anticancer activities of QA root extract and its regulatory pathways in two human breast cancer cell lines (MCF-7 and SUM159). Materials and methodsDried QA root barks were extracted by ethanol and used to treat human breast cancer MCF-7 and SUM159 cells with varying concentrations. The CCK-8 assay, Hoechst 33342 staining assay and wound healing assay were used to detect the cell proliferation, apoptotic cell morphology, and cell migration in each group, respectively. Caspase 3 activity assay kit was used to determine caspase 3 activity. Western blot was used to measure proteins expression level in apoptosis and autophagy pathways (Bcl-W, caspase 3, Beclin1, LC3 and Atg5). LC-MS was performed to determine the chemical components in QA root extract. ResultsCCK-8 assay showed that QA root extract significantly inhibited cell viability and proliferation in breast cancer cells by a concentration-dependent manner. Cell wound healing assay indicated that it had high suppression ability on cell migration both in MCF-7 and SUM159 cells. QA root extract treatment induced the morphological and nuclear structural changes in breast cancer cells including rounded appearance and shrunken nucleus with several nuclear body fragments. Western blot indicated that QA root extract induced mitochondria-mediated apoptosis by up-regulating caspase 3 and down-regulating Bcl-W. Moreover, QA root extract up-regulated Beclin1 and Atg5, and activated LC3 in two human breast cancer cell lines. LC-MS results showed that QA root extract contains high content of bioactive compounds like coumarins and derivatives, prenol lipids, flavonoids and tannins. ConclusionsQA root extract inhibited cell proliferation and migration in MCF-7 and SUM159 cells, and it also induced cell morphology changes and regulated mitochondria-mediated apoptotic cell death and autophagic cell death.

TRPM2 channel in oxidative stress-induced mitochondrial dysfunction and apoptotic cell death.

Mitochondria, conserved intracellular organelles best known as the powerhouse of cells for generating ATP, play an important role in apoptosis. Oxidative stress can induce mitochondrial dysfunction and activate mitochondria-mediated apoptotic cell death. TRPM2 is a Ca2+-permeable cation channel that is activated by pathologically relevant concentrations of reactive oxygen species (ROS) and one of its well-recognized roles is to confer susceptibility to ROS-induced cell death. Increasing evidence from recent studies supports TRPM2 channel-mediated cell death as an important cellular mechanism linking miscellaneous oxidative stress-inducing pathological factors to associated diseased conditions. In this chapter, we will discuss the role of the TRPM2 channel in neurons in the brain and pancreatic β-cells in mediating mitochondrial dysfunction and cell death, focusing mainly on apoptotic cell death, that are induced by pathological stimuli implicated in the pathogenesis of neurodegenerative diseases, ischemic stroke and diabetes.

Astaxanthin: The possible effects of this carotenoid on disease, inflammation and aging control. A meta-analysis

Astaxanthin (ATX), a red pigment that belongs to the xanthophyll subclass of carotenoids, has a strong antioxidant ability and can eliminate singlet oxygen (O2-) as well as hydrogen peroxide (H2O2) and lipid peroxidation. ATX can also prevent mitochondrial dysfunction by permeating and co-localizing within the mitochondria and inhibit the release of cytochrome c resulting from mitochondrial permeabilization and, thus, prevent mitochondrial-mediated apoptotic cell death. Due to its antioxidant capacity and modulating properties of cell signaling, ATX exhibits a variety of beneficial biological activities among them protection against UV damage, anti-inflammatory and immunomodulatory activity, metabolic syndrome (MS) relief, cardioprotective effects, antidiabetic activity, prevention of neuronal damage, anti-aging and anticancer activity. The aim of the present study was to evaluate what has been published about ATX in PubMed/Medline between 2020-2021. The results were distributed in four Tables as follows: Table 1-Publication types; Table 2- Proposal for evaluating the article in vivo; Table 3- Cells markers used in clinical studies in vivo; Table 4- Astaxanthin in human clinical trial. We could observe that the interest of the scientific community has been growing in relation to the benefits of ATX. The results presented in the articles evaluated in this meta-analysis showed us that AXT is already a reality as an option in treatments for various diseases, including glaucoma, heart and vascular injury, type 2 diabetes and fatty liver. We conclude that ATX may not only be a promising nutraceutical as an ally to alternative treatments of the pathologies mentioned above, but also as a powerful prophylactic in elderly individuals in prevention of diseases associated with aging.

Pd(II) and Rh(III) Complexes with Isoquinoline Derivatives Induced Mitochondria-Mediated Apoptotic and Autophagic Cell Death in HepG2 Cells

Nonplatinum metal complexes of [Pd(L1)Cl2] (C1), [Rh(L1)Cl3(DMSO)] (C2), [Pd(L2)Cl2] (C3), and [Rh(L3)Cl3(DMSO)] (C4) with isoquinoline derivatives have been prepared and characterized. C1–C4 exhib...

Coenzyme Q10 mitigates ionizing radiation-induced testicular damage in rats through inhibition of oxidative stress and mitochondria-mediated apoptotic cell death

Radiotherapy is a common treatment modality for cancer patients; however, its use is limited by decreasing the probability of fertility in male cancer survivors. Therefore, this study aimed to define the capability of coenzyme Q10 (CoQ10), a potent stimulator of mitochondrial function, in attenuating ionizing radiation (IR)-induced spermatogenesis impairments. Male Sprague Dawley rats were exposed to a single dose of ϒ-rays (10 Gy) and/or treated with CoQ10 (10 mg/kg, orally, for 2 consecutive weeks). IR mediated irregular seminiferous tubules, which were emerged with typical morphological characteristics of apoptosis, and nuclear condensation, while CoQ10 significantly preserved the testicular structure and maintained spermatogenesis, which was displayed by higher levels of serum estradiol and testosterone. CoQ10 remarkably augmented sperm count, motility, and viability while diminished the rate of sperm-defects relatively to their counterparts after IR exposure. CoQ10 modulations in reproductive parameters were underpinned by attenuating IR-induced oxidative stress as evidenced by decreasing lipid peroxidation and increasing the antioxidant enzymes glutathione peroxidase and glutathione-s-transferase activities, and glutathione level. Supporting the involvement of CoQ10 in the anti-apoptotic response, the reduced mRNA expression levels of p53, Puma, and Bax accompanied by the increased Bcl-2 mRNA expression were observed. Subsequently, CoQ10 ameliorated the mitochondria dependent apoptotic pathway through diminishing Bax/Bcl-2 ratio, caspase-3 protein expression, and DNA fragmentation in testes of irradiated rats. Taken together, our findings showed that CoQ10 conserved against IR-induced steroidogenesis disruption through subsiding mitochondria-mediated oxidative stress injury in germinal cells.

Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells.

Malignant melanoma is the most life-threatening neoplasm of the skin. Despite the increase in incidence, melanoma is becoming more resistant to current therapeutic agents. The bioactive compound frugoside has been recently reported to inhibit growth when used in various cancer cells. However, this effect has not been demonstrated in melanoma. Here, we found that frugoside inhibited the rate of reduction of hyperoxidized peroxiredoxins (Prxs) by downregulating sulfiredoxin (Srx) expression. Furthermore, frugoside increased the accumulation of sulfinic Prxs and reactive oxygen species (ROS) and stimulated p-p38 activation, resulting in the mitochondria-mediated death of M14 and A375 human melanoma cells. The mitochondria-mediated cell death induced by frugoside was inhibited by the overexpression of Srx and antioxidants, such as N-acetyl cysteine and diphenyleneiodonium. In addition, we observed that frugoside inhibited tumor growth without toxicity through a M14 xenograft animal model. Taken together, our findings reveal that frugoside exhibits a novel antitumor effect based on a ROS-mediated cell death in melanoma cells, which may have therapeutic implications.

Two novel platinum(II) complexes with sorafenib and regorafenib: Synthesis, structural characterization, and evaluation of in vitro antitumor activity

Two new Pt(II) complexes with sorafenib (SRFN) and regorafenib (RGFN), having the general formulae [Pt(SRFN)(DMSO)Cl2] (SRFN-Pt) and [Pt(RGFN)(DMSO)Cl2] (RGFN-Pt), were prepared and characterized by ESI-MS, IR, UV–Vis spectroscopy, elemental analyses, and 1H and 13C NMR, respectively. The anticancer activities of SRFN-Pt and RGFN-Pt were evaluated by MTT assay with NCI-H460 (human non-small cell lung cancer NCI-H460 cell line), SK-OV-3 (ovarian cancer cell line), SK-OV-3/DDP (cisplatin-resistant SK-OV-3 cell line), T-24 (human bladder cancer cell line), HeLa (cervical cancer cell line), A549/DDP (cisplatin-resistant A549/DDP non-small cell lung cancer cell line) cancer cells and in the normal HL-7702 cells. The results suggested that SRFN-Pt and RGFN-Pt were more effective against the A549/DDP tumor cells (IC50 = 1.18 ± 0.15 μM and 0.13 ± 0.03 μM) than SRFN (45.03 ± 0.79 μM), RGFN (40.11 ± 2.15 μM), and cisplatin (97.63 ± 1.06 μM), respectively, and RGFN-Pt was more effective than SRFN-Pt. In addition, SRFN-Pt and RGFN-Pt induced G2/M and S phase arrest. Cytotoxic mechanism studies revealed that SRFN-Pt and RGFN-Pt triggered mitochondria-mediated apoptotic cell death at low concentration. RGFN-Pt exhibited obvious priority on the in vitro antitumor activity than SRFN-Pt, which should be undoubtedly correlated with the key roles of the fluoro substituted groups in the RGFN ligand of RGFN-Pt. The in vitro anti-tumor activity studies suggested that RGFN-Pt pointed to a new direction in developing Pt(II) drugs as anti-cancer agent.

Cotreatment with sorafenib and oleanolic acid induces reactive oxygen species-dependent and mitochondrial-mediated apoptotic cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most common liver malignancy, and the lack of effective chemotherapies underlines the need for novel therapeutic approaches for this disease. Recently, we discovered a novel synergistic induction of cell death by combining sorafenib, the only routinely used palliative chemotherapeutic agent, and the triterpenoid oleanolic acid (OA). However, the underlying mechanisms of action have remained obscure. Here, we report that sorafenib and OA acted in concert to trigger mitochondria-mediated apoptotic cell death, which is dependent on reactive oxygen species (ROS). Sorafenib/OA cotreatment significantly increased ROS production, which was prevented by the ROS scavengers α-tocopherol and MnTBAP. Importantly, rescue experiments showed that ROS were required for sorafenib/OA-induced apoptosis as ROS scavengers protected HCC cells against cell death. In addition, sorafenib and OA cotreatment cooperated to decrease myeloid cell leukaemia-1 expression and to activate Bak, two events that were prevented by ROS scavengers. Bak activation was accompanied by the loss of mitochondrial membrane potential, followed by PARP cleavage, DNA fragmentation and, finally, apoptotic cell death in HCC cells. By providing new insights into the molecular regulation of sorafenib/OA-mediated and ROS-dependent cell death, our study contributes toward the development of novel treatment strategies to overcome sorafenib resistance in HCC.

A novel splicing variant of small nucleolar RNA host gene 4 is a podocyte-selective non-coding RNA upregulated in response to puromycin aminonucleoside-induced podocyte injury.

Podocytes are terminally differentiated cells that function as the glomerular filtration barrier in the kidney, and podocyte injury leads to serious proteinuria and podocyte leakage into urine. Recent studies have demonstrated that the number of urinary podocytes is correlated with the progression of glomerular diseases. Therefore, urinary podocytes may serve as an indicator of podocyte injury. In this study, to explore podocyte injury-related genes, we performed comprehensive transcriptome analysis of primary rat podocytes cultured in the presence or absence of puromycin aminonucleoside (PAN), an agent commonly used to induce podocyte injury. RNA-seq revealed that a transcript containing the intronic sequence of small nucleolar RNA host gene 4 (Snhg4) was expressed in podocytes and upregulated by PAN. RT-qPCR analysis demonstrated that this transcript, but not Snhg4, was selectively expressed in podocytes. Therefore, we designated the novel transcript Snhg4-pod. 5'- and 3'-RACE experiments revealed that Snhg4-pod is a novel splice variant of Snhg4 lacking a poly(A) tail. PAN induced Snhg4-pod expression in podocytes in a dose-dependent manner along with their mitochondria-mediated apoptotic cell death. Further, Snhg4-pod was detected in urinary sediments from PAN-induced nephrotic rats. Our findings suggest that Snhg4-pod may serve as a novel marker for the diagnosis of glomerular injury.

The role of formation of pyrrole-ATP synthase subunit beta adduct in pyrrolizidine alkaloid-induced hepatotoxicity.

Pyrrolizidine alkaloids (PAs) are one of the most significant groups of hepatotoxic phytotoxins. It is well-studied that metabolic activation of PAs generates reactive pyrrolic metabolites that rapidly bind to cellular proteins to form pyrrole-protein adducts leading to hepatotoxicity. Pyrrole-protein adducts all contain an identical core pyrrole moiety regardless of structures of the different PAs; however, the proteins forming pyrrole-protein adducts are largely unknown. The present study revealed that ATP synthase subunit beta (ATP5B), a critical subunit of mitochondrial ATP synthase, was a protein bound to the reactive pyrrolic metabolites forming pyrrole-ATP5B adduct. Using both anti-ATP5B antibody and our prepared anti-pyrrole-protein antibody, pyrrole-ATP5B adduct was identified in the liver of rats, hepatic sinusoidal endothelial cells, and HepaRG hepatocytes treated with retrorsine, a well-studied representative hepatotoxic PA. HepaRG cells were then used to further explore the consequence of pyrrole-ATP5B adduct formation. After treatment with retrorsine, significant amounts of pyrrole-ATP5B adduct were formed in HepaRG cells, resulting in remarkably reduced ATP synthase activity and intracellular ATP level. Subsequently, mitochondrial membrane potential and respiration were reduced, leading to mitochondria-mediated apoptotic cell death. Moreover, pre-treatment of HepaRG cells with a mitochondrial membrane permeability transition pore inhibitor significantly reduced retrorsine-induced toxicity, further revealing that mitochondrial dysfunction caused by pyrrole-ATP5B adduct formation significantly contributed to PA intoxication. Our findings for the first time identified ATP5B as a protein covalently bound to the reactive pyrrolic metabolites of PAs to form pyrrole-ATP5B adduct, which impairs mitochondrial function and significantly contributes to PA-induced hepatotoxicity.

TUBP1 protein lead to mitochondria-mediated apoptotic cell death in Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is a cancer of cotton which affects cotton quality and yield in China. In our previous study, a novel anti-Verticillium dahliae protein TUBP1 was obtained from Bacillus axarquiensis. Then, we have systematically studied the anti-V. dahliae activity and the pore formation action of TUBP1 protein on V. dahliae membrane. In present study, we provide detailed whether TUBP1 protein induced mitochondrial damaged and mitochondria-mediated apoptotic cell death in V. dahliae. In V. dahliae cells exposed to the TUBP1 protein, the mitochondrial dehydrogenases, F0F1-ATPase, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) activities were reduced and reactive oxygen species (ROS), which is a major cause of apoptosis, were increased. The results demonstrated that mitochondria dysfunction and ROS-induced oxidative stress caused the release of apoptotic factors. The following cellular changes, which are characteristic of apoptosis, were measured including mitochondrial membrane potential (MMP), Cytochrome c (Cyt C) release, metacaspase activation, phosphatidylserine (PS) exposure, and DNA condensation and fragmentation. The results showed that an important feature of apoptosis, MMP, was caused by ROS. Significantly, cyt c was released, which is a factor in metacaspase activity after treatment with the TUBP1 protein. Number of stained cells with activated intracellular metacaspases exposed to TUBP1 protein was increased in a concentration-dependent manner. We also showed that in the early and late stages of apoptosis, the effects of the TUBP1 protein were mediated by PS and DNA fragmentation and condensation in the plasma membrane, respectively. There turned out that the TUBP1 protein led to mitochondria-mediated apoptotic cell death in V.dahliae. The results of this investigation indicated that TUBP1 stain or protein is a potent candidate against V.dahliae infections in crop species.

Silica - A trace geogenic element with emerging nephrotoxic potential

Silica is a trace-geogenic compound with limited-bioavailability. It inflicts health-perils like pulmonary-silicosis and chronic kidney disease (CKD), when available via anthropogenic-disturbances. Amidst silica-imposed pathologies, pulmonary toxicological-mechanisms are well-described, ignoring the renal-pathophysiological mechanisms. Hence, the present-study aimed to elucidate cellular-cum-molecular toxicological-mechanisms underlying silica-induced renal-pathology in-vitro.Various toxicity-assessments were used to study effects of silica on the physiological-functions of HK-cells (human-kidney proximal-tubular cells - the toxin's prime target) on chronic (1–7 days) sub-toxic (80 mg/L) and toxic (100–120 mg/L) dosing.Results depicted that silica triggered dose-cum-time dependent cytotoxicity/cell-death (MTT-assay) that significantly increased on long-term dosing with ≥100 mg/L silica; establishing the nephrotoxic-potential of this dose. Contrarily, insignificant cell-death on sub-toxic (80 mg/L) dosing was attributed to rapid intracellular toxin-clearance at lower-doses preventing toxic-effects. The proximal-tubular (HK-cells) cytotoxicity was found to be primarily mediated by silica-triggered incessant oxidative-stress (elevated ROS).·This enhanced ROS inflicted severe inflammation and subsequent fibrosis, evident from increased pro-inflammatory-cum-fibrogenic cytokines generation (IL-1β, IL-2, IL-6, TNF-α and TGF-β). Simultaneously, ROS induced persistent DNA-damage (Comet-assay) that stimulated G2/M arrest for p53-mediated damage-repair, aided by checkpoint-promoter (Chk1) activation and mitotic-inducers (i.e. Cdc-25, Cdk1, cyclinB1) inhibition. However, DNA-injuries surpassed the cellular-repair, which provoked the p53-gene to induce mitochondrial-mediated apoptotic cell-death via activation of Bax, cytochrome-c and caspase-cascade (9/3). This persistent apoptotic cell-death and simultaneous incessant inflammation culminated in the development of tubular-atrophy and fibrosis, the major pathological-manifestations of CKD.These findings provided novel-insights into the pathological-mechanisms (cellular and molecular) of silica-induced CKD, inflicted on chronic toxic-dosing (≥100 mg/L).Thereby, encouraging the development of therapeutic-strategies (e.g. anti-oxidant treatment) for specific molecular-targets (e.g. ROS) to retard silica-induced CKD-progression, for reduction in the global-CKD burden.

Greensporone C, a Freshwater Fungal Secondary Metabolite Induces Mitochondrial-Mediated Apoptotic Cell Death in Leukemic Cell Lines.

Therapeutic agents used in the treatment of cancer are known to develop resistance against cancer cells. Hence, there is a continuing need to investigate novel agents for the treatment and management of cancer. Antitumor activity of greensporone C (GC), a new resorcylic acid lactone isolated from an organic extract of a culture of a Halenospora sp. freshwater fungus, was subjected for screening against a panel of leukemic cell lines (K562, U937, and AR320). In all the three cell lines, cell proliferation was inhibited in dose-dependent fashion. GC further arrested the cells in SubG0 phase in dose-dependent manner. Annexin V/PI dual staining data confirmed apoptotic death of treated K562 and U937 leukemic cells. Treatment with GC suppressed constitutively phosphorylated AKT and downregulated expression of inhibitor of apoptotic proteins XIAP, cIAP-1, and cIAP-2. In summation to this, GC-treated leukemic cells upregulated protein expression of pro-apoptotic proteins, Bax with concomitant decrease in expression of anti-apoptotic proteins including Bcl-2 and Bcl-xL. Upregulation of Bax was associated with cytochrome c release which was confirmed from the collapse of mitochondrial membrane. Released cytochrome c further activated caspase cascade which in turn initiated apoptosis process. Anticancer activity of this isolated fungal compound GC was potentiated via stimulating production of reactive oxygen species (ROS) along with depletion of reduced glutathione (GSH) levels in K562 and U937 leukemic cells. Pretreatment of these cells with N-acetyl cysteine prevented GC-induced depletion of reduced GSH level and mitochondrial-caspase-induced apoptosis. Altogether, our data show that GC modulates the apoptotic response of human leukemic cells and raises the possibility of its use as a novel therapeutic strategy for hematological malignancies.

Mixed-ligand Cu(II) hydrazone complexes designed to enhance anticancer activity

The ligand quantity, ligand type, and coordination geometry have important influences on the anticancer activity of metal-based complexes. On the basis of the structures of previously reported 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H2O 1a, [Cu(L2)Cl]·H2O 2a, and [Cu(L2)NO3]·H2O 3a), we subsequently designed, developed, and characterized a series of corresponding 1:1:1 Cu(II)/ligand/co-ligand complexes ([Cu(L1)(Py)Cl]·H2O 1b, [Cu(L2)(Py)Cl] 2b, and [Cu(L2)(Py)NO3] 3b), where L1 = (E)-N'-(2-hydroxybenzylidene)acetohydrazide, L2 = (E)-N'-(2- hydroxybenzylidene)benzohydrazide, and Py = pyridine. All six Cu(II) complexes were assessed for their in vitro anticancer properties against a panel of human cancer cells, including cisplatin-resistant A549cisR cell lines. Interestingly, we observed that the 1:1:1 Cu/ligand/co-ligand mixed-ligand Cu(II) complexes exhibited higher anticancer activity than the corresponding 1:1 Cu(II)/ligand complexes. In particular, the 1:1:1 Cu(II)/ligand/co-ligand complex 3b displayed the greatest toxicity toward several cancer cells with better IC50 (1.12–3.77 μM) than cisplatin. Further mechanistic explorations showed that the 3b complex induced DNA damage, thus resulting in mitochondria-mediated apoptotic cell death. Furthermore, the 3b complex displayed pronounced cytostatic effects in the MCF-7 3D spheroid model.