Effectiveness of Gold Nanorods of Different Sizes in Photothermal Therapy to Eliminate Melanoma and Glioblastoma Cells.

Gold nanorods are the most commonly used nanoparticles in photothermal therapy for cancer treatment due to their high efficiency in converting light into heat. This study aimed to investigate the efficacy of gold nanorods of different sizes (large and small) in eliminating two types of cancer cell: melanoma and glioblastoma cells. After establishing the optimal concentration of nanoparticles and determining the appropriate time and power of laser irradiation, photothermal therapy was applied to melanoma and glioblastoma cells, resulting in the highly efficient elimination of both cell types. The efficiency of the PTT was evaluated using several methods, including biochemical analysis, fluorescence microscopy, and flow cytometry. The dehydrogenase activity, as well as calcein-propidium iodide and Annexin V staining, were employed to determine the cell viability and the type of cell death triggered by the PTT. The melanoma cells exhibited greater resistance to photothermal therapy, but this resistance was overcome by irradiating cells at physiological temperatures. Our findings revealed that the predominant cell-death pathway activated by the photothermal therapy mediated by gold nanorods was apoptosis. This is advantageous as the presence of apoptotic cells can stimulate antitumoral immunity in vivo. Considering the high efficacy of these gold nanorods in photothermal therapy, large nanoparticles could be useful for biofunctionalization purposes. Large nanorods offer a greater surface area for attaching biomolecules, thereby promoting high sensitivity and specificity in recognizing target cancer cells. Additionally, large nanoparticles could also be beneficial for theranostic applications, involving both therapy and diagnosis, due to their superior detection sensitivity.

Hydroxytyrosol-Donepezil Hybrids Play a Protective Role in an In Vitro Induced Alzheimer's Disease Model and in Neuronal Differentiated Human SH-SY5Y Neuroblastoma Cells.

Alzheimer's disease (AD) is the most common neurodegenerative pathology among progressive dementias, and it is characterized by the accumulation in the brain of extracellular aggregates of beta-amyloid proteins and neurofibrillary intracellular tangles consisting of τ-hyperphosphorylated proteins. Under normal conditions, beta-amyloid peptides exert important trophic and antioxidant roles, while their massive presence leads to a cascade of events culminating in the onset of AD. The fibrils of beta-amyloid proteins are formed by the process of fibrillogenesis that, starting from individual monomers of beta-amyloid, can generate polymers of this protein, constituting the hypothesis of the "amyloid cascade". To date, due to the lack of pharmacological treatment for AD without toxic side effects, chemical research is directed towards the realization of hybrid compounds that can act as an adjuvant in the treatment of this neurodegenerative pathology. The hybrid compounds used in this work include moieties of a hydroxytyrosol, a nitrohydroxytyrosol, a tyrosol, and a homovanillyl alcohol bound to the N-benzylpiperidine moiety of donepezil, the main drug used in AD. Previous experiments have shown different properties of these hybrids, including low toxicity and antioxidant and chelating activities. The purpose of this work was to test the effects of hybrid compounds mixed with Aß1-40 to induce fibrillogenesis and mimic AD pathogenesis. This condition has been studied both in test tubes and by an in vitro model of neuronal differentiated human SH-SY5Y neuroblastoma cells. The results obtained from test tube experiments showed that some hybrids inhibit the activity of the enzymes AChE, BuChE, and BACE-1. Cell experiments suggested that hybrids could inhibit fibrillogenesis, negatively modulating caspase-3. They were also shown to exert antioxidant effects, and the acetylated hybrids were found to be more functional and efficient than nonacetylated forms.

Revisited Metabolic Control and Reprogramming Cancers by Means of the Warburg Effect in Tumor Cells.

Aerobic glycolysis is an emerging hallmark of many human cancers, as cancer cells are defined as a "metabolically abnormal system". Carbohydrates are metabolically reprogrammed by its metabolizing and catabolizing enzymes in such abnormal cancer cells. Normal cells acquire their energy from oxidative phosphorylation, while cancer cells acquire their energy from oxidative glycolysis, known as the "Warburg effect". Energy-metabolic differences are easily found in the growth, invasion, immune escape and anti-tumor drug resistance of cancer cells. The glycolysis pathway is carried out in multiple enzymatic steps and yields two pyruvate molecules from one glucose (Glc) molecule by orchestral reaction of enzymes. Uncontrolled glycolysis or abnormally activated glycolysis is easily observed in the metabolism of cancer cells with enhanced levels of glycolytic proteins and enzymatic activities. In the "Warburg effect", tumor cells utilize energy supplied from lactic acid-based fermentative glycolysis operated by glycolysis-specific enzymes of hexokinase (HK), keto-HK-A, Glc-6-phosphate isomerase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, phosphofructokinase (PFK), phosphor-Glc isomerase (PGI), fructose-bisphosphate aldolase, phosphoglycerate (PG) kinase (PGK)1, triose phosphate isomerase, PG mutase (PGAM), glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase isozyme type M2 (PKM2), pyruvate dehydrogenase (PDH), PDH kinase and lactate dehydrogenase. They are related to glycolytic flux. The key enzymes involved in glycolysis are directly linked to oncogenesis and drug resistance. Among the metabolic enzymes, PKM2, PGK1, HK, keto-HK-A and nucleoside diphosphate kinase also have protein kinase activities. Because glycolysis-generated energy is not enough, the cancer cell-favored glycolysis to produce low ATP level seems to be non-efficient for cancer growth and self-protection. Thus, the Warburg effect is still an attractive phenomenon to understand the metabolic glycolysis favored in cancer. If the basic properties of the Warburg effect, including genetic mutations and signaling shifts are considered, anti-cancer therapeutic targets can be raised. Specific therapeutics targeting metabolic enzymes in aerobic glycolysis and hypoxic microenvironments have been developed to kill tumor cells. The present review deals with the tumor-specific Warburg effect with the revisited viewpoint of recent progress.

Neuroprotective effect of the somatostatin receptor 5 agonist L-817,818 on retinal ganglion cells in experimental glaucoma.

Somatostatin plays important roles in modulating neuronal functions by activating the five specific G-protein coupled receptors (sst1-sst5). Previous studies have demonstrated that sst5 were expressed in retinal ganglion cells (RGCs) and sst5 agonist attenuated the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-induced retinal neurotoxicity. In this study, we investigated effects and underlying mechanisms of the sst5 agonist L-817,818 on RGC injury induced by elevated intraocular pressure (COH) in experimental glaucoma. Our results showed that intraperitoneal administration of L-817,818 significantly reduced RGC loss and decreased the number of terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive RGCs in COH retinas, suggesting that L-817,818 may attenuate RGC apoptosis. Consistently, in COH retinas with L-817,818 administration, both the down-regulated mRNA and protein levels of anti-apoptotic Bcl-2 and the up-regulated mRNA and protein levels of pro-apoptotic Bax were partially reversed. L-817,818 administration downregulated the expression of apoptosis-related proteins caspase-9 and caspase-3 in COH retinas. In addition, L-817,818 administration reduced the concentrations of reactive oxygen species/reactive nitrogen species and malondialdehyde, and ameliorated the functions of mitochondrial respiratory chain complex (MRCC). Our results imply that administration of the sst5 agonist L-817,818 reduces RGC loss in COH rats through decreasing RGC apoptosis, which is mediated by regulating Bcl-2/Bax balance, reducing oxidative stress and rescuing activities of MRCC. Activation of sst5 may provide neuroprotective roles for RGCs in glaucoma.

Green synthesized-zinc oxide nanoparticles, the strong apoptosis inducer as an exclusive antitumor agent in murine breast tumor model and human breast cancer cell lines (MCF7).

PURPOSE: In the present study, we aimed to synthesize and investigate the impact of zinc oxide nanoparticle (ZnONPs) on both human and murine breast cancer cell lines and define their untoxic concentrations (IC50 ) to clarify their apoptotic properties and introduce them as the anticancer agents. MATERIALS AND METHODS: The in vitro study was initiated by ZnONPs green synthesizing process applying the Cucumis melo inodorus rough shell extract, and verified by the transmission electron microscope, scanning electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction analysis. In following, the human (Michigan Cancer Foundation-7 [MCF7]) and murine (TUBO) breast cancer cell lines were cultured for taking the time and dose-dependent treatment planes by ZnONPs. Also, MCF7 cell cultures were treated by three different doses of ZnoNPs (8, 4, and 2 µg/mL) separately and prepared for genes expression (Cas-3 and Cas-8) analysis using real-time quantitative PCR method. The in vivo initiated by providing the 39 murine breast cancer models, then they were injected intraperitoneally with different doses of ZnONPs (75, 50, and 25 mg/kg) treatments. Then their collected biopsies were stained by hematoxylin and eosin to evaluate their breast cancer tissue morphology and compare with Tamoxifen anticancer properties. RESULTS: The in vitro study results demonstrate a significant correlation among the expression of Cas-3 and Cas-8 genes with increasing ZnONPs concentrations. The results of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assays for the treated cancer cell lines (MCF7 and TUBO) detected a significant negative correlation among the ZnONPs concentrations and the viability of the cells. CONCLUSION: Unlike the majority of resent studies, we found the ZnONPs as a powerful apoptosis inducer in the human cell line (MCF7) and murine (TUBO cell line and cancer model).

BMN 673 (talazoparib): A potent PARP inhibitor for triple negative breast cancer with different genetic profile.

The objective of the present study was to elucidate the effect of BMN 673 (talozoparib) on BRCA1 mutant (HCC1937) and wild-type (MDA-MB-231) triple negative breast cancer (TNBC). The in vitro cytotoxicity results indicated that BMN 673 had considerable inhibitory effects on HCC1937 and MDA-MB-231 cell lines by inducing apoptosis, multicaspase activity, G2/M arrest, and altering the expression levels of apoptosis-related genes (P < 0.01). Additionally, BMN 673 indicated no toxicity on MCF-10A control cells until a certain concentration and incubation time. However, BMN 673, a novel and selective poly ADP ribose polymerase inhibitor, was more potent in TNBC cells bearing BRCA1 mutant than those with wild-type BRCA1. In conclusion, our study, for the first time, demonstrated a molecular mechanism of the induction of apoptosis by BMN 673 in TNBC with different genetic profile. However, further investigations regarding the exact molecular mechanisms underlying BMN 673-inducing apoptotic death and gene-cell line associations are required.

Producing the sour cherry pit oil nanoemulsion and evaluation of its anti-cancer effects on both breast cancer murine model and MCF-7 cell line.

Aims: The sour cherry pit oil (SCPO) displays the potent anti-inflammatory, and antioxidant activities. In the present study, we have produced the SCPO nanoemulsion (SCPO-NE) to evaluate their anticancer impacts on breast cancer comparing with its un-processed oil. Methods: We employed an ultrasonication method to formulate the stable SCPO-NE. Their size, stability, and morphology were measured. Then, their cytotoxic impacts and apoptotic activity were checked on MCF7 breast cancer cells and compared with the normal Human foreskin fibroblasts (HFF). Finally, their anti-tumour effect was studied on murine breast cancer model (inoculated with TUBO cancer cells). Results: The results indicated the 36.5 nm stable SCPO-NE significantly decreased the MCF7 cells viability comparing with normal HFF cells, and reduced the tumour size in the murine model. Conclusion: We suggest that SCPO-NEs are able to efficiently inhibit breast cancer progression in both MCF7 cells and murine breast cancer model through apoptotic death induction.

Targeting Nrf2-mediated heme oxygenase-1 enhances non-thermal plasma-induced cell death in non-small-cell lung cancer A549 cells.

Non-thermal plasma (NTP) treatment has been proposed as a potential approach for cancer therapy for killing cancer cells via generation of reactive oxygen species (ROS). As an antioxidant protein, Heme oxygenase-1 (HO-1) has been known to protect cells against oxidative stress. In this paper, we investigated the role of HO-1 activation in NTP-induced apoptosis in A549 cells. Distinctly increased ROS production and apoptosis were observed after NTP exposure. NTP exposure induced HO-1 expression in a dose- and time-dependent manner via activating the translocation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) from cytoplasm to nucleus. Furthermore, inhibiting HO-1 activation with its specific inhibitor, ZnPP, increased "killing" effect of NTP. Knocking down HO-1 or Nrf2 with the special siRNA also led to elevated ROS level and enhanced NTP-induced cell death. In addition, the c-JUN N-terminal kinase (JNK) signaling pathway was shown to be involved in NTP-induced HO-1 expression. Interestingly, a higher resistance to NTP exposure of A549 cell compared to H1299 and H322 cells was found to be linked to its higher basal level of HO-1 expression. These findings revealed that HO-1 could be considered as a potential target to improve the effect of NTP in cancer therapy.

Magnetic nanoparticle-mediated hyperthermia therapy induces tumour growth inhibition by apoptosis and Hsp90/AKT modulation.

PURPOSE: We have evaluated the hyperthermia efficacy of oleic acid-functionalised Fe(3)O(4) magnetic nanoparticles (MN-OA) under in vivo conditions and elucidated the underlying mechanism of tumour growth inhibition. MATERIALS AND METHODS: The efficacy and mechanism of tumour growth inhibition by MN-OA-mediated magnetic hyperthermia therapy (MHT) was evaluated in a murine fibrosarcoma tumour model (WEHI-164) using techniques such as TUNEL assay, Western blotting (WB), immunofluorescence (IF) staining and histopathological examination. In addition, bio-distribution of MN-OA in tumour/other target organs and its effect on normal organ function were studied by Prussian blue staining and serum biochemical analysis, respectively. RESULTS: MN-OA-induced MHT resulted in significant inhibition of tumour growth as determined by measurement of tumour volume, as well as by in vivo imaging of tumour derived from luciferase-transfected WEHI-164 cells. Histopathology analysis showed presence of severe apoptosis and reduced tumour cells proliferation, which was further confirmed by TUNEL assay, reduced expression of Ki-67 and enhanced level of cleaved caspase-3, in tumours treated with MHT. Moreover, expression of heat stress marker, Hsp90 and its client protein, AKT/PKB was reduced by ∼50 and 80%, respectively, in tumours treated with MHT as studied by WB and IF staining. Serum analysis suggested insignificant toxicity of MN-OA (in terms of liver and kidney function), which was further correlated with minimal accumulation of MN-OA in target organs. CONCLUSIONS: These results suggest the involvement of apoptosis and Hsp90/AKT modulation in MN-OA-mediated MHT-induced tumour growth inhibition.

The anticancer impact of folate-linked ZnO-decorated bovine serum albumin/silibinin nanoparticles on human pancreatic, breast, lung, and colon cancers.

In the current study, we aimed to design an individual hybrid silibinin nano-delivery system consisting of ZnO and BSA components to study its antioxidant activity and apoptotic potential on human pancreatic, breast, lung, and colon cancer cell lines. The folate-linked ZnO-decorated bovine serum albumin/silibinin nanoparticles (FZBS-NP) were synthesized and characterized by FTIR, FESEM, DLS, and zeta potential analysis. The FZBS-NP's cytotoxicity was evaluated by measuring the cancer cells' (MCF-7, A549, HT-29, and Panc) viability. Moreover, the apoptotic potential of the nanoparticles was studied by conducting several analyses including AO/PI and DAPI cell staining analysis, apoptotic gene expression profile (BAX, BCL2, and Caspase-8) preparation, and FITC Annexin V/PI flow cytometry. Finally, both antioxidant assays (ABTS and DPPH) were utilized to analyze the FZBS-NPs' antioxidant activities. The 152-nm FZBS-NP significantly induced the selective apoptotic death on the MCF-7, A549, HT-29, Panc, and Huvec cancer cells by increasing the SubG1 cell population following the increased treatment concentrations of FZBS-NP. Moreover, the FZBS-NPs exhibited powerful antioxidant activity. The BSA component of the FZBS-NPs delivery system improves the ability of the nanoparticles to gradually release silibinin and ZnO near the cancer cells. On the other hand, considering the powerful antioxidant activity of FZBS-NP, they have the potential to selectively induce apoptosis in human colon and breast cancer cells and protect normal types, which makes it an efficient safe anticancer compound. However, to verify the FZBS-NP anti-cancer efficiency further cancer and normal cell lines are required to measure several types of apoptotic gene expression.

DPEP Inhibits Cancer Cell Glucose Uptake, Glycolysis and Survival by Upregulating Tumor Suppressor TXNIP.

We have designed cell-penetrating peptides that target the leucine zipper transcription factors ATF5, CEBPB and CEBPD and that promote apoptotic death of a wide range of cancer cell types, but not normal cells, in vitro and in vivo. Though such peptides have the potential for clinical application, their mechanisms of action are not fully understood. Here, we show that one such peptide, Dpep, compromises glucose uptake and glycolysis in a cell context-dependent manner (in about two-thirds of cancer lines assessed). These actions are dependent on induction of tumor suppressor TXNIP (thioredoxin-interacting protein) mRNA and protein. Knockdown studies show that TXNIP significantly contributes to apoptotic death in those cancer cells in which it is induced by Dpep. The metabolic actions of Dpep on glycolysis led us to explore combinations of Dpep with clinically approved drugs metformin and atovaquone that inhibit oxidative phosphorylation and that are in trials for cancer treatment. Dpep showed additive to synergistic activities in all lines tested. In summary, we find that Dpep induces TXNIP in a cell context-dependent manner that in turn suppresses glucose uptake and glycolysis and contributes to apoptotic death of a range of cancer cells.

Monte Carlo simulation for assessing absorbed dose effects of low-dose ß-radiation (90Sr/90Y) on cytotoxicity and apoptotic death in K562 cells.

Objective: Most studies suggest that dose-specific radiation regimens are essential for optimal induction of cancer cell response. This study focused on determining ß-radiation-absorbed dose (rad) effects on the cell viability, cytotoxicity, hypersensitivity, and cell death of K562 cells using experimental methods and Monte Carlo simulation (MCS). Materials and Methods: The K562 cells were cultured and irradiated with ß-particles emitted from a strontium source in vitro, with the estimated daily activity of 1.238 µCi. The treated cells were radiated at least three times every day for 3 consecutive days. The cell viability and apoptosis were investigated in treated cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA electrophoresis, Hoechst dye, and inverted microscope. The average absorbed doses were obtained by MCS (MCNPX code). To verify simulation and experimental results, we used a Geiger-Muller counter and estimated a scaling factor. Results: The cytotoxic effects and cell death were induced in the treated groups via rad in a time-dependent manner. The highest apoptotic and cytotoxic effects were observed in cells after irradiation with ß-particles for 120 min per day in 3 consecutive days. rads were determined using MCNPX code and cell survival rates were significantly reduced during irradiation periods. No significant hyper-radiosensitivity was found based on experimental and theoretical results. Conclusion: Despite the difficult calculation of the rad in the target cells and the scant information in this field, fortunately we have achieved significant theoretical data consistent with the experimental results. Our findings also introduced MCS as a better choice for evaluating of rad effects under different cellular conditions with high accuracy.

Response Mechanisms of Invertebrates to Bacillus thuringiensis and Its Pesticidal Proteins.

Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives is bioinsecticides based on Bacillus thuringiensis This entomopathogenic bacterium produces different protein types which are toxic to several insect, mite, and nematode species. Currently, insecticidal proteins belonging to the Cry and Vip3 groups are widely used to control insect pests both in formulated sprays and in transgenic crops. However, the benefits of B. thuringiensis-based products are threatened by insect resistance evolution. Numerous studies have highlighted that mutations in genes coding for surrogate receptors are responsible for conferring resistance to B. thuringiensis Nevertheless, other mechanisms may also contribute to the reduction of the effectiveness of B. thuringiensis-based products for managing insect pests and even to the acquisition of resistance. Here, we review the relevant literature reporting how invertebrates (mainly insects and Caenorhabditis elegans) respond to exposure to B. thuringiensis as either whole bacteria, spores, and/or its pesticidal proteins.

Role of Death Receptors-associated Lipid Rafts in Oxaliplatin-induced Death Mode Regulation of HepG2 Cells.

BACKGROUND/AIM: We previously showed that oxaliplatin induces necrotic-like cell death in hepatocarcinomas, and combination with ursodexoycholic acid (UDCA) significantly shifts the necrotic-like death to apoptosis. Since cell death mode is crucial on inflammatory responses and chemotherapeutic efficacy, the mechanism underlying determination of cell death mode by UDCA was investigated in this study. MATERIALS AND METHODS: Apoptosis or necrosis was determined by apoptotic body formation, caspase-8 activity, LDH release and PI inclusion. The involvement of lipid rafts and death receptors was examined by rafts fractionation, confocal microscopy and gene silencing assays. RESULTS: UDCA combination enhanced recruitment of death receptors and adaptors into cholesterol-enriched lipid rafts, and induced a stronger raft clustering. Lipid raft disruption decreased the UDCA/oxaliplatin-mediated apoptosis and increased necrotic-like death. CONCLUSION: UDCA promotes lipid raft localization of multiple death receptors, thereby contributing to a shift of cell death mode from oxaliplatin-induced necrotic death to apoptosis in HepG2 cells.

Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent.

APO866 is a small molecule drug that specifically inhibits nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Although, the antitumor activity of APO866 on various types of cancer models has been reported, information regarding mechanisms by which APO866 exerts its cytotoxic effects is not well defined. Here we show that APO866 induces a strong, time-dependent increase in highly reactive ROS, nitric oxide, cytosolic/mitochondrial superoxide anions and hydrogen peroxide. We provide evidence that APO866-mediated ROS production is modulated by PARP1 and triggers cell death through mitochondria depolarization and ATP loss. Genetic or pharmacologic inhibition of PARP1 prevented hydrogen peroxide accumulation, caspase activation, mitochondria depolarization, ATP loss and abrogates APO866-induced cell death, suggesting that the integrity of PARP1 status is required for cell death. Conversely, PARP1 activating drugs enhanced the anti-leukemia activity of APO866 Collectively, our studies show that APO866 induces ROS/RNS productions, which mediate its anti-leukemia effect. These results support testing new combinatorial strategies to enhance the antitumor activities of APO866.

GmDAD1, a Conserved Defender Against Cell Death 1 (DAD1) From Soybean, Positively Regulates Plant Resistance Against Phytophthora Pathogens.

Initially identified as a mammalian apoptosis suppressor, defender against apoptotic death 1 (DAD1) protein has conserved plant orthologs acting as negative regulators of cell death. The potential roles and action mechanisms of plant DADs in resistance against Phytophthora pathogens are still unknown. Here, we cloned GmDAD1 from soybean and performed functional dissection. GmDAD1 expression can be induced by Phytophthora sojae infection in both compatible and incompatible soybean varieties. By manipulating GmDAD1 expression in soybean hairy roots, we showed that GmDAD1 transcript accumulations are positively correlated with plant resistance levels against P. sojae. Heterologous expression of GmDAD1 in Nicotiana benthamiana enhanced its resistance to Phytophthora parasitica. NbDAD1 from N. benthamiana was shown to have similar role in conferring Phytophthora resistance. As an endoplasmic reticulum (ER)-localized protein, GmDAD1 was demonstrated to be involved in ER stress signaling and to affect the expression of multiple defense-related genes. Taken together, our findings reveal that GmDAD1 plays a critical role in defense against Phytophthora pathogens and might participate in the ER stress signaling pathway. The defense-associated characteristic of GmDAD1 makes it a valuable working target for breeding Phytophthora resistant soybean varieties.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner.

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment.

Non-apoptotic cell death associated with perturbations of macropinocytosis.

Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed "methuosis," from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death.

Calcium ions (Ca(2+)) are indispensable for the physiology of organisms and the molecular regulation of cells. We observed that CGK733, a synthetic chemical substance, induced non-apoptotic cell death and stimulated reversible calcium sequestration by vesicles in pancreatic cancer cells. The endoplasmic reticulum (ER) stress eukaryotic translation initiation factor 2-alpha kinase 3/C/EBP homologous protein (PERK/CHOP) signaling pathway was shown to be activated by treatment with CGK733. Ionomycin, an ER stress drug and calcium ionophore, can activate PERK/CHOP signaling and accelerate CGK733-induced calcium sequestration. Knockdown of CHOP diminished CGK733-induced vesicular calcium sequestration, but had no effects on the cell death. Proteomic analysis demonstrated that the ER-located calcium-binding proteins, calumenin and protein S100-A11, were altered in CGK733-treated cells compared to non-treated controls. Our study reveals that CGK733-induced intracellular calcium sequestration is correlated with the PERK/CHOP signaling pathway and may also be involved in the dysregulations of calcium-binding proteins.