Primary Mechanism Of Cell Death Research Articles

Essential Oil of Hymenocrater longiflorus Benth. as a Pro-Apoptotic Agent in Human Breast Cancer Cells

Background: Breast cancer is one of the most common cancers in women. Current treatments, such as chemotherapy and radiation therapy, often involve numerous side effects. Consequently, it is essential to discover new anticancer compounds that are safer and more effective. Objectives: The aim of this research is to investigate the anticancer effects of Hymenocrater longiflorus Benth. essential oil. Methods: The anticancer properties of H. longiflorus Benth. essential oil were investigated on MDA-MB-231 and MCF-7 breast cancer cells using MTT assay, double staining with acridine orange/ethidium bromide, and flow cytometry. The essential oil of H. longiflorus Benth. was analyzed using gas chromatography-mass spectrometry (GC-MS). Results: The essential oil significantly reduced the viability of breast cancer cells depending on the concentration (dose) and duration. Results from double fluorescent staining and flow cytometric analysis also indicated that apoptosis is the primary mechanism of cell death. Non-targeted GC-MS metabolomic data analysis of the essential oil revealed the presence of various anticancer compounds, including α-pinene, linalool, and caryophyllene, in the essential oil. Conclusions: In conclusion, the essential oil of H. longiflorus Benth. is a promising source of anticancer metabolites that can be further evaluated for drug therapy or as a co-treatment with conventional chemotherapeutic agents.

Ischemia-Reperfusion Increases TRPM7 Expression in Mouse Retinas.

Ischemia is the main cause of cell death in retinal diseases such as vascular occlusions, diabetic retinopathy, glaucoma, or retinopathy of prematurity. Although excitotoxicity is considered the primary mechanism of cell death during an ischemic event, antagonists of glutamatergic receptors have been unsuccessful in clinical trials with patients suffering ischemia or stroke. Our main purpose was to analyze if the transient receptor potential channel 7 (TRPM7) could contribute to retinal dysfunction in retinal pathologies associated with ischemia. By using an experimental model of acute retinal ischemia, we analyzed the changes in retinal function by electroretinography and the changes in retinal morphology by optical coherence tomography (OCT) and OCT-angiography (OCTA). Immunohistochemistry was performed to assess the pattern of TRPM7 and its expression level in the retina. Our results show that ischemia elicited a decrease in retinal responsiveness to light stimuli along with reactive gliosis and a significant increase in the expression of TRPM7 in Müller cells. TRPM7 could emerge as a new drug target to be explored in retinal pathologies associated with ischemia.

Syntheses, characterization of Imidazo[4,5-f][1,10]phenanthroline derivative and As (III) complex, In vitro evaluation, the determine of apoptosis mechanism; theoretical and quantum studies

In the present work, a novel ligand, (4-amino-3-methoxybenzaldehyde)-1H-imidazo[4,5-f][1,10] phenanthroline (AMIP) based on the formation of an imidazole ring and [As(AMIP)](Cl)]Cl2 was synthesized and characterized through Fourier-transform infrared (FT-IR) spectroscopy. In addition, the anticancer activity of [As(AMIP)](Cl)]Cl2 complex was tested against human acute promyelocytic leukemia cells (HL-60) and granulocytic differentiation of human promyelocytic leukemia cells (NB4). The MTT results showed IC50 values of 7.31 ± 15 and 8.4 ± 62 μM for As (III) complex against NB4 and HL-60, respectively. In a study on both cell lines, As (III) reduced gene expression Bcl-2 and Bcl-xl, increased Bax and Bad, and activated caspase-3, resulting in apoptosis. The dye Hoechst 33342 showed increased apoptosis induction in the NB4 cell line than in the HL-60 cell line. BrdU test exhibited an inhibitory effect on DNA synthesis. The protein levels of caspase-3 were enhanced in a dose-dependent manner in both HL-60 and NB-4 cell lines. Based on the Annexin V-FITC/PI assay, apoptosis was the primary mechanism of cell death. The real-time polymerase chain reaction (real-time PCR) results demonstrated that the expression level of Atg 12, Atg 7, Lc 3, and Beclin-1 genes, antiapoptotic genes including Bcl-2 and Bcl-xl, and apoptotic genes containing BAD and BAX was improved. Furthermore, the hemolysis assay showed the anti-hemolytic activity of the complex. Moreover, quantum chemical calculations were utilized to optimize all structures, and the output results were employed to study molecular docking of compounds with caspase-3 tethered to irreversible inhibitor (PDB ID: 1NMS) and DNA with sequence d(CCGTCGACGG) (PDB ID:423D). The results showed that As (III) complex had the lowest-energy conformation, suggesting the optimal interaction.

The Effect of Infant Gastric Digestion on Human Maternal Milk Cells.

Human breast milk contains a variety of cell types that have potential roles in infant immunity and development. One challenge associates with defining the purpose(s) of milk cells in the infant is a poor understanding of the effect of digestion on cell fate. This study first demonstrates that milk cell death occurs after gastric digestion in mice. Then flow cytometry and RT-PCR are used to understand the mechanism of human milk cell death and quantify live cell types before and after simulated gastric digestion. This study finds that digestion in simulated gastric fluid for 30 min reduces cell viability from 72% to 27%, with most cell death is caused by the acidic pH. The primary mechanism of cell death is caspase-mediated apoptosis. The non-cellular components of milk offer only mild protection against cell death from stomach acid. Gastric digestion does not select for a specific resilient cell population to survive-most cell types die in equal proportions in the gastric environment. Taken together, these results provide a foundation with which to understand the fate of human breast milk cells in the infant's intestine and beyond.

Abstract 1832: CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian cancer

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1832.

Abstract 1291: The novel c-MYC inhibitor IDP-121 exhibits strong anti-myeloma effect

Abstract Multiple myeloma (MM) is a blood malignancy having its origin in differentiated B cells.1 The overall survival of MM patients has clearly improved with the use of autologous stem cell transplantation and the more recent introduction of novel therapeutic strategies. However, for the majority of patients, the disease will eventually relapse even after good initial response and remains incurable. The uncontrolled activation of c-Myc due to translocation, mutation or alteration of upstream signalling pathways is a common characteristic of many cancers including MM.2 No compound targeting c-Myc is yet used in the clinic, mostly due to its intra-nuclear location and its intrinsically disordered nature, that renders small molecule or antibody therapy development problematic. Here we describe the anti-myeloma effect of IDP-121, a stapled peptide specifically designed to target c-MYC protein (see Abstract 1). IDP-121 reduced viability of a large set of MM cell lines, with apoptosis as the primary mechanism of cell death. Short contact time was sufficient to exert its anti-myeloma effect reflecting rapid cellular uptake. Similar reduction of cell viability was observed ex vivo by multiparameter flow cytometry in primary cells derived from MM patients. Interestingly, normal lymphocytes from these same patients, were largely preserved from IDP-121-induced apoptosis, suggesting its selectivity and the presence of a therapeutic window. IDP-121 was able to rapidly disrupt the c-MYC/MAX complex in cells as confirmed by immune-precipitation and FRET experiments. The complex disruption was accompanied by the reduction of whole c-MYC protein, probably trigging monomer degradation. No effect on c-Myc gene expression levels was observed, consistent with a direct c-MYC protein inhibitor. IDP-121 exposure led to c-MYC nuclear depletion, with consequent reduction of the binding of the transcription factor complex to DNA. In vivo, IDP-121 shows significant reduction of tumor growth in a subcutaneous plasmacytoma and a disseminated xenograft model of MM. The HPLC-MS and IHC analyses of tumor tissue after treatment confirms effective tumoral tissue distribution and target engagement, with significant reduction of c-MYC protein and cell proliferation in vivo. Finally, and importantly, preliminary combination experiments highlight that IDP-121 potentiated the activity of MM standards of care such as bortezomib and dexamethasone. The results presented here suggest that IDP-121 could be a first-in-class inhibitor of c-MYC in MM. The drug-like properties and efficacy also in solid tumors of IDP-121 have been demonstrated in vivo (see Abstracts 3). In addition, IDP-121 has progressed through GLP toxicology studies without evidence of major systemic toxicity, allowing for the first viable Myc-targeted therapy to enter Phase 1 clinical trials in 2021. 1Bergsagel PL, et al. J Am Soc Clin Oncol. 2005; 23:6333–8.2Holien T., et al. Blood. 2012; 120: 2450–3. Citation Format: Patryk Krzeminski, Lorena González-Méndez, Laura San Segundo, Pedro Mogollón, Andrea Diaz, Susana Hernández-García, Macarena Algarín, Montserrat Martín-Sánchez, Luis Cochete, Teresa Paino, Santiago Esteban, Laura Nevola, Norma C. Gutiérrez, Mª-Victoria Mateos, Mercedes Garayoa, Enrique M. Ocio. The novel c-MYC inhibitor IDP-121 exhibits strong anti-myeloma effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1291.

Mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors

Accumulating data indicates that some cancer treatments can restore anticancer immunosurveillance through the induction of tumor immunogenic cell death (ICD). Nanosecond pulsed electric fields (nsPEF) have been shown to efficiently ablate melanoma tumors. In this study we investigated the mechanisms and immunogenicity of nsPEF-induced cell death in B16F10 melanoma tumors. Our data show that in vitro nsPEF (20–200, 200-ns pulses, 7 kV/cm, 2 Hz) caused a rapid dose-dependent cell death which was not accompanied by caspase activation or PARP cleavage. The lack of nsPEF-induced apoptosis was confirmed in vivo in B16F10 tumors. NsPEF also failed to trigger ICD-linked responses such as necroptosis and autophagy. Our results point at necrosis as the primary mechanism of cell death induced by nsPEF in B16F10 cells. We finally compared the antitumor immunity in animals treated with nsPEF (750, 200-ns, 25 kV/cm, 2 Hz) with animals were tumors were surgically removed. Compared to the naïve group where all animals developed tumors, nsPEF and surgery protected 33% (6/18) and 28.6% (4/14) of the animals, respectively. Our data suggest that, under our experimental conditions, the local ablation by nsPEF restored but did not boost the natural antitumor immunity which stays dormant in the tumor-bearing host.

Potent anti-proliferative, pro-apoptotic activity of the Maytenus royleanus extract against prostate cancer cells: evidence in in-vitro and in-vivo models.

Prostate cancer is a leading of cause of cancer related death in men. Despite intensive investment in improving early diagnosis, it often escapes timely detection. Mortality remains high in advanced stage prostate cancer where palliative care remains the only option. Effective strategies are therefore needed to prevent the occurrence and progression of the disease. Plant-derived compounds have been an important source of several clinically useful anti-cancer agents and offer an attractive approach against prostate cancer. We previously showed that the methanol extract of Maytenus royleanus (MEM) leaves and its fractions possess significant antioxidant activity with therapeutic potential against free-radical associated damages. The present study evaluated the anti-proliferative activity of MEM in the prostate cancer model system. Analysis of MEM and its various fractions revealed the presence of triterpenoids, flavonoids and tannins, conjugated to one or more polar groups and carbohydrate moieties. Further studies against known standards established the existence of caffeic acid and quercetin 3-rhamnoside in varying concentration in different MEM fractions. Time course analysis of MEM treated prostate cancer cells indicated significant decrease in cell viability, assessed by MTT and clonogenic survival assays. This was accompanied by G2 phase arrest of cell cycle, downregulation of cyclin/cdk network and increase in cdk inhibitors. MEM treated cells exhibited cleavage of Caspase-3 and PARP, and modulation of apoptotic proteins, establishing apoptosis as the primary mechanism of cell death. Notably MEM suppressed AR/PSA signaling both in prostate cancer cell cultures and in the in vivo model. Intraperitoneal injection of MEM (1.25 and 2.5mg/ animal) to athymic nude mice implanted with androgen sensitive CWR22Rν1 cells showed significant inhibition in tumor growth and decreased serum PSA levels reciprocating in vitro findings. Taken together, our data suggest that MEM may be explored further for its potential therapeutic effects against prostate cancer progression in humans.

Lithocholic acid feeding results in direct hepato-toxicity independent of neutrophil function in mice

Lithocholic acid (LCA) supplementation in the diet results in intrahepatic cholestasis and bile infarcts. Previously we showed that an innate immune response is critical for cholestatic liver injury in the bile duct ligated mice. Thus, the purpose of this study was to investigate the role of neutrophils in the mechanism of liver injury caused by feeding mice a diet containing LCA. C57BL/6 mice were given control or 1% LCA containing diet for 24–96h and then examined for parameters of hepatotoxicity. Plasma ALT levels were significantly increased by 48h after LCA feeding, which correlated with both neutrophil recruitment to the liver and upregulation of numerous pro-inflammatory genes. The injury was confirmed by histology. Deficiency in intercellular adhesion molecule-1 (ICAM-1) expression or inhibition of neutrophil function failed to protect against the injury. Bile acid levels were quantified in plasma and bile of LCA-fed mice after 48 and 96h. Only the observed biliary levels of taurochenodeoxycholic acid and potentially tauro-LCA caused direct cytotoxicity in mouse hepatocytes. These data support the conclusion that neutrophil recruitment occurs after the onset of bile acid-induced necrosis in LCA-fed animals, and is not a primary mechanism of cell death when cholestasis occurs through accumulation of hydrophobic bile acids.

Evaluation of the molecular mechanisms of a palladium(II) saccharinate complex with terpyridine as an anticancer agent

Metal-based compounds represent promising anticancer therapeutic agents. In this study, the mechanism of action of a novel metal-based drug, a palladium(II) (Pd) complex ([PdCl(terpy)](sac)·2H2O, terpy=2,2':6',2''-terpyridine and sac=saccharinate), was elucidated. The tested compound induced cytotoxicity in nine different human cancer cell lines that originated from various organs, suggesting a broad spectrum of activity. The IC50 values were significantly higher for noncancerous cells when compared with cancer cells. We found that cells treated with the Pd(II) complex exhibited increased caspase 3/7 activities and condensed/fragmented nuclei, as demonstrated by nuclear staining and DNA laddering. Morphological features, such as cellular shrinkage and blebbing, were also observed, indicating that apoptosis was the primary mechanism of cell death. Pd(II) treatment induced DNA double-stranded breaks both in vitro and in vivo, potentially accounting for the source of stress in these cells. Although caspase 3/7 activities were elevated after Pd(II) treatment, silencing or using inhibitors of caspase 3 did not block apoptosis. Other molecules that could potentially play a role in Pd(II)-induced apoptosis, such as p53 and Bax, were also tested using silencing technology. However, none of these proteins were essential for cell death, indicating either that these molecules do not participate in Pd(II)-induced apoptosis or that other pathways were activated in their absence. Hence, this new molecule might represent a promising anticancer agent that exhibits cytotoxicity in p53-mutant, Bax-mutant, and/or caspase 3-mutant cancer cells.

Mechanisms of Antibacterial Activity of MgO: Non‐ROS Mediated Toxicity of MgO Nanoparticles Towards Escherichia coli

The toxicity of metal oxide nanomaterials and their antimicrobial activity is attracting increasing attention. Among these materials, MgO is particularly interesting as a low cost, environmentally-friendly material. The toxicity of MgO, similar to other metal oxide nanomaterials, is commonly attributed to the production of reactive oxygen species (ROS). We investigated the toxicity of three different MgO nanoparticle samples, and clearly demonstrated robust toxicity towards Escherichia coli bacterial cells in the absence of ROS production for two MgO nanoparticle samples. Proteomics data also clearly demonstrate the absence of oxidative stress and indicate that the primary mechanism of cell death is related to the cell membrane damage, which does not appear to be due to lipid peroxidation.

Antioxidant and Pro-Apoptotic Effects of Marine-Derived, Multi-Mineral Aquamin Supplemented with a Pine Bark Extract, Enzogenol, and a Green Tea Extract, Sunphenon

A high dietary intake of polyphenols has been associated with a decreased risk of cardiovascular disease and cancer, attributed in part to their antioxidant activity and pro-apoptotic effects. Aquamin is a multi-mineral algal extract that enhances bone mineralization, relieves osteoarthritis, and aids digestion; however, Aquamin has not demonstrated antioxidant activity. In the present study, Aquamin was supplemented with 8% Enzogenol, a pine bark extract with a high phenolic content, and 2% Sunphenon, a green tea extract that also has a high phenolic content to produce a mixed product (A:E:S). The antioxidant activity of A:E:S was compared with that of its constituent compounds and also with catechin and epigallocatechin by measuring total phenol content, ferric-reducing antioxidant potential, and 2,2-diphenyl-2-picrylhydrazyl hydrate. The cytotoxic and apoptotic effects of the compounds were also measured in the U937, human monocytic blood cell line. A:E:S demonstrated an antioxidant activity that was equal to that of the compounds used in its preparation. Aquamin was not cytotoxic in the U937 cell line; however, A:E:S was cytotoxic and the primary mechanism of cell death was apoptosis. The biological effects of Aquamin were enhanced by supplementation with Enzogenol and Sunphenon to include antioxidant effects and the ability to induce apoptosis in U937 cells.

Neuronal necrosis and spreading death in a Drosophila genetic model

Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death.

Reovirus Activates a Caspase-Independent Cell Death Pathway

ABSTRACTVirus-induced apoptosis is thought to be the primary mechanism of cell death following reovirus infection. Induction of cell death following reovirus infection is initiated by the incoming viral capsid proteins during cell entry and occurs via NF-κB-dependent activation of classical apoptotic pathways. Prototype reovirus strain T3D displays a higher cell-killing potential than strain T1L. To investigate how signaling pathways initiated by T3D and T1L differ, we methodically analyzed cell death pathways activated by these two viruses in L929 cells. We found that T3D activates NF-κB, initiator caspases, and effector caspases to a significantly greater extent than T1L. Surprisingly, blockade of NF-κB or caspases did not affect T3D-induced cell death. Cell death following T3D infection resulted in a reduction in cellular ATP levels and was sensitive to inhibition of the kinase activity of receptor interacting protein 1 (RIP1). Furthermore, membranes of T3D-infected cells were compromised. Based on the dispensability of caspases, a requirement for RIP1 kinase function, and the physiological status of infected cells, we conclude that reovirus can also induce an alternate, necrotic form of cell death described as necroptosis. We also found that induction of necroptosis requires synthesis of viral RNA or proteins, a step distinct from that necessary for the induction of apoptosis. Thus, our studies reveal that two different events in the reovirus replication cycle can injure host cells by distinct mechanisms.

Efficacy of substituted 9-aminoacridine derivatives in small cell lung cancer

Topoisomerase II (TopoII) plays a critical role in the processes of replication, transcription, and decantenation in the cell and is an important chemotherapeutic target in the treatment of small cell lung cancer (SCLC). Current treatment strategies for SCLC employ the use of topoII poisons which stabilize the topoII-DNA transient covalent complex, inducing double stranded DNA damage and cellular death via apoptosis in cancer cells. Despite their effectiveness the topoII poisons are known to induce secondary malignancies in a small population of patients, stimulating the search for new compounds with less toxicity. Recently a small library of substituted 9-aminoacridine derivatives was discovered that displayed topoII catalytic inhibitory properties. In this work we assess their ability to inhibit proliferation and induce cellular death in SCLC. The results indicate effective inhibition of cellular proliferation at EC(50) values in the low μM range. Western blot analysis of p62/LC3 levels, the AKT/mTOR pathway, and the ERK1/2 pathway indicate that autophagy is occurring as the primary mechanism of cell death; furthermore, the Guava Nexin and caspase 3/7 activation assays indicate that apoptosis does not occur. While it is unlikely that the active concentration of these compounds could be achieved in vivo, they show great promise for the use and effectiveness of acridine derivatives in the treatment of SCLC in the future.

Kinetic studies of the effects of Temodal and quercetin on astrocytoma cells

The aim of the present study was to investigate the kinetics of the effects exerted by Temodal and quercetin on the survival of the human astrocytoma MOGGCCM cell line. Our results indicate that quercetin was toxic and induced necrosis, whereas Temodal induced autophagy-mediated cell death most effectively. The amount of cell death directly correlated with drug concentration and length of exposure. During combined administration of both drugs, Temodal attenuated the cytotoxic effects of quercetin. Combinations of both drugs were effective in inducing programmed cell death, but the type of cell death was concentration-dependent. Coadministration of Temodal (100µM) with a low quercetin concentration (5µM) resulted in a very significant induction of autophagy; however, after treatment with quercetin at a higher concentration (30µM), apoptosis became the primary mechanism of cell death. The sequence of drug administration was also important. The highest number of dead cells was observed after simultaneous administration of both drugs or after pre-incubation with Temodal followed by treatment with quercetin. Apoptosis was identified through activation of the mitochondrial pathway including cleavage of caspase-3 and release of cytochrome c. Autophagy was identified through increased levels of LC3II. Our results indicate that Temodal and quercetin are synergistic inducers of programmed cell death, better together than applied separately. This drug combination appears to be a potent and promising therapeutic relevant to the treatment of gliomas.

Kavalactones Yangonin and Methysticin Induce Apoptosis in Human Hepatocytes (HepG2) In Vitro

While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ~ 40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved.

Mitomycin C Inhibits Ribosomal RNA: A NOVEL CYTOTOXIC MECHANISM FOR BIOREDUCTIVE DRUGS

Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds.

Characterization of cell death events induced by anti-neoplastic drugs cisplatin, paclitaxel and 5-fluorouracil on human hepatoma cell lines: Possible mechanisms of cell resistance

Two different hepatoma cell lines were incubated for 48 h with chemotherapeutic drugs cisplatin, paclitaxel and 5-FU to determine their ability to induce cytotoxicity and DNA fragmentation as well as to modify the expression of some cell death-related genes that could be involved in the resistance to therapy. We observed that cisplatin and paclitaxel induced cytotoxicity, but significant differences between both cell lines, were found only in the case of paclitaxel. At 48 h, apoptosis was clearly present in Hep3B cells treated with cisplatin and HepG2 cells treated with paclitaxel. 5-FU induced cytotoxicity in both cell lines but only at higher concentrations than the other two drugs, triggering apoptosis and necrosis in HepG2 cells and only necrosis in Hep3B. When a time course was performed for the first 8 h of treatment to elucidate the initial mechanism of cell death responsible for DNA fragmentation, we observed that 5-FU in Hep3B, and cisplatin in both cell lines, induces primary necrosis, whereas at the concentration tested here, paclitaxel clearly triggers apoptosis in both cell lines. HepG2 cells were weakly sensitive to 5-FU in the first 8 h of treatment, so the primary mechanism of cell death was not clear, but results seem to indicate that it could be apoptosis. At 48 h, Bax was not up-regulated with any of the treatments, whereas cisplatin was able to induce Bcl-xL down-regulation in both cell lines. Treatment with 5-FU also down-regulated Bcl-xL in HepG2 cells. We also measured variations in the expression of survivin, an inhibitor of apoptosis that has also been involved in mitototic catastrophe. Hep3B cells seem to show an increase in protein levels with all treatments. Exposure to paclitaxel resulted in the highest effect. In the case of HepG2 cells, there was a decrease in survivin expression when cells were treated with 5FU and paclitaxel, both treatments showing complete loss of the protein. Using an antibody that recognizes unprocessed caspase-3, we observed that the enzyme was assumingly activated in HepG2 cells treated with 5FU and paclitaxel, but only weakly after treatment with cisplatin. Hep3B cells did not show activation since the levels of the pro-enzyme remained the same as that in the control. In conclusion, the three drugs tested in this study could induce cell death, with paclitaxel being more effective inducing apoptosis. 5FU was only effective at high doses and its mechanism seems to be primarily related to necrosis in Hep3B and probably apoptosis in HepG2. Cisplatin mechanism of cell death is probably mediated by the decrease in anti-apoptotic protein Bcl-xL whereas paclitaxel and 5FU are decreasing the apoptosis inhibitor survivin. According to pro-enzyme levels, caspase-3 was only activated in HepG2 cells, whereas in the case of Hep3B cells the mechanisms of toxicity appear to be caspase-3-independent at the time and concentrations tested in this study. The resistance of Hep3B cells to death induced by chemotherapy could be related to an increase in the expression of IAP survivin, which can decrease cell response to the treatment or even switch the type of death from apoptosis to another kind, making therapy less efficient.

Disease and cell death in white syndrome of Acroporid corals on the Great Barrier Reef

White syndromes (WS) are among the most prevalent coral diseases, and are responsible for reef demise on the Great Barrier Reef. The disease manifests as a clear differentiation between tissue and exposed skeleton and results in rapid tissue loss. Fluorescence in situ hybridisation (FISH) was used in conjunction with histology and transmission electron microscopy (TEM) to investigate bacterial communities and cell death associated with WS. No evidence of bacterial communities or microbial association (using six bacterial probes, TEM and histopathology) was evident within the lesion or adjacent tissues, despite the presence of dense possible secondary invaders in the exposed skeletal regions. Despite widespread reference to necrosis in coral disease literature, there was no evidence of necrosis in any WS lesion or the adjacent tissues in this study. However, in situ end labelling, light microscopy and TEM of WS and healthy coral tissue sections showed evidence of extensive programmed cell death (PCD) exclusively in WS. This study provides the first evidence of intrinsic or PCD as a primary mechanism of cell death in WS, and may provide some explanation for the failure to isolate pathogens from over 80% of identified coral diseases, many of which show similar lesion patterns and WS characteristics.