Activation Of Intrinsic Apoptotic Pathway Research Articles

Alpha-Pinene As a Novel Therapeutic Agent for T-Cell Tumors

T-cell acute leukemia and lymphoma have poor prognoses, necessitating the development of novel therapeutic agents. In this study, we investigated the antitumor activity of α-pinene, a monoterpene compound, against T-cell tumors. We evaluated the effects of α-pinene on cell growth inhibition in vitro using the murine T-cell tumor cell line EL-4 and the human-derived T-cell tumor cell line Molt-4. In addition, we assessed the effects of limonene, another monoterpene with reported tumor-suppressive effects, as a comparative agent. Both α-pinene and limonene demonstrated concentration- and time-dependent inhibition of cell growth in both the EL-4 and Molt-4 cell lines (Figure 1). Moreover, α-pinene exhibited greater potency than limonene in inhibiting the growth of various hematologic malignancies. Importantly, α-pinene and limonene had minimal effects on the growth of normal murine spleen T cells. Further investigation of the mechanisms of action of α-pinene revealed its ability to induce apoptosis and cell cycle arrest in EL-4 and Molt-4 cells. Transcriptomic profiling of α-pinene-treated EL-4 cells using RNA-seq identified differentially expressed genes associated with cellular damage and mitochondrial dysfunction. Increased intracellular ROS levels and mitochondrial impairment were observed in the T-cell tumors treated with α-pinene. The activation of intrinsic apoptotic pathways involving EGR1, p53, BCL-2, and BAX was found to contribute to α-pinene-induced apoptosis in T-cell tumors. Moreover, α-pinene inhibited the NF-κB signaling pathway, resulting in the reduced nuclear translocation of NF-κB p65 and decreased total intracellular NF-κB p65 levels in EL-4 cells. Additionally, we discovered that α-pinene induced ferroptosis, a newly identified programmed cell death process, in EL-4 cells through lipid peroxidation and iron accumulation. Activation of the system x c−/GSH/GPX4 axis and upregulation of iron transporters, such as Slc39a8 and TfR1, were observed in α-pinene-induced ferroptosis. Treatment with the ferroptosis inhibitor Fer-1 partially reversed the tumor-inhibiting effect of α-pinene in EL-4 cells. Finally, we administrated α-pinene to mice subcutaneously injected with luciferase-expressing EL-4 to evaluate the efficacy of α-pinene in vivo. The tumor growth was significantly inhibited by α-pinene treatment (vehicle: 1055 ± 101 mm 3; α-pinene: 701 ± 82 mm 3, p < 0.01, Figure 2) without adverse effects on body weight or behavior. Immunohistochemistry analyses revealed a significant increase of CD8 + T-cells (vehicle: 8.1 ± 2.7 cells / field of view; α-pinene: 43.1 ± 12.2 cells / field of view , p < 0.05) and NK1.1 + cells (vehicle: 5.1 ± 1.0 cells / field of view ; α-pinene: 41.1 ± 9.8 cells / field of view , p < 0.01) in the tumors, suggesting that α-pinene may have an indirect antitumor effect by recruiting immune cells into tumors. Overall, our findings demonstrate the antitumor activity of α-pinene against T-cell tumors through the induction of apoptosis, cell cycle arrest, and ferroptosis. α-Pinene shows promise as a potential therapeutic agent for T-cell tumors and warrants further investigation for its clinical application.

Sidestream Smoke Extracts from Harm-Reduction and Conventional Camel Cigarettes Inhibit Osteogenic Differentiation via Oxidative Stress and Differential Activation of intrinsic Apoptotic Pathways.

Epidemiological studies suggest cigarette smoking as a probable environmental factor for a variety of congenital anomalies, including low bone mass, increased fracture risk and poor skeletal health. Human and animal in vitro models have confirmed hypomineralization of differentiating cell lines with sidestream smoke being more harmful to developing cells than mainstream smoke. Furthermore, first reports are emerging to suggest a differential impact of conventional versus harm-reduction tobacco products on bone tissue as it develops in the embryo or in vitro. To gather first insight into the molecular mechanism of such differences, we assessed the effect of sidestream smoke solutions from Camel (conventional) and Camel Blue (harm-reduction) cigarettes using a human embryonic stem cell osteogenic differentiation model. Sidestream smoke from the conventional Camel cigarettes concentration-dependently inhibited in vitro calcification triggered by high levels of mitochondrially generated oxidative stress, loss of mitochondrial membrane potential, and reduced ATP production. Camel sidestream smoke also induced DNA damage and caspase 9-dependent apoptosis. Camel Blue-exposed cells, in contrast, invoked only intermediate levels of reactive oxygen species insufficient to activate caspase 3/7. Despite the absence of apoptotic gene activation, damage to the mitochondrial phenotype was still noted concomitant with activation of an anti-inflammatory gene signature and inhibited mineralization. Collectively, the presented findings in differentiating pluripotent stem cells imply that embryos may exhibit low bone mineral density if exposed to environmental smoke during development.

Overcoming acquired resistance to third-generation EGFR inhibitors by targeting activation of intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation, or both.

Treatment of EGFR-mutant non-small cell lung cancer (NSCLC) with mutation-selective third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib has achieved remarkable success in the clinic. However, the immediate challenge is the emergence of acquired resistance, limiting the long-term remission of patients. This study suggests a novel strategy to overcome acquired resistance to osimertinib and other third-generation EGFR-TKIs through directly targeting the intrinsic apoptotic pathway. We found that osimertinib, when combined with Mcl-1 inhibition or Bax activation, synergistically decreased the survival of different osimertinib-resistant cell lines, enhanced the induction of intrinsic apoptosis, and inhibited the growth of osimertinib-resistant tumor in vivo. Interestingly, the triple-combination of osimertinib with Mcl-1 inhibition and Bax activation exhibited the most potent activity in decreasing the survival and inducing apoptosis of osimertinib-resistant cells and in suppressing the growth of osimertinib-resistant tumors. These effects were associated with increased activation of the intrinsic apoptotic pathway evidenced by augmented mitochondrial cytochrome C and Smac release. Hence, this study convincingly demonstrates a novel strategy for overcoming acquired resistance to osimertinib and other 3rd generation EGFR-TKIs by targeting activation of the intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation or both, warranting further clinical validation of this strategy.

Alkylaminophenol and GPR17 Agonist for Glioblastoma Therapy: A Combinational Approach for Enhanced Cell Death Activity.

Drug resistance and tumor heterogeneity limits the therapeutic efficacy in treating glioblastoma, an aggressive infiltrative type of brain tumor. GBM cells develops resistance against chemotherapeutic agent, temozolomide (TMZ), which leads to the failure in treatment strategies. This enduring challenge of GBM drug resistance could be rational by combinatorial targeted therapy. Here, we evaluated the combinatorial effect of phenolic compound (2-(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), GPR17 agonist 2-({5-[3-(Morpholine-4-sulfonyl)phenyl]-4-[4-(trifluoromethoxy)phenyl]-4H-1,2,4-triazol-3-yl}sulfanyl)-N-[4-(propan-2-yl)phenyl]acetamide (T0510.3657 or T0) with the frontline drug, TMZ, on the inhibition of GBM cells. Mesenchymal cell lines derived from patients’ tumors, MMK1 and JK2 were treated with the combination of THTMP + T0, THTMP + TMZ and T0 + TMZ. Cellular migration, invasion and clonogenicity assays were performed to check the migratory behavior and the ability to form colony of GBM cells. Mitochondrial membrane permeability (MMP) assay and intracellular calcium, [Ca2+]i, assay was done to comprehend the mechanism of apoptosis. Role of apoptosis-related signaling molecules was analyzed in the induction of programmed cell death. In vivo validation in the xenograft models further validates the preclinical efficacy of the combinatorial drug. GBM cells exert better synergistic effect when exposed to the cytotoxic concentration of THTMP + T0, than other combinations. It also inhibited tumor cell proliferation, migration, invasion, colony-forming ability and cell cycle progression in S phase, better than the other combinations. Moreover, the combination of THTMP + T0 profoundly increased the [Ca2+]i, reactive oxygen species in a time-dependent manner, thus affecting MMP and leading to apoptosis. The activation of intrinsic apoptotic pathway was regulated by the expression of Bcl-2, cleaved caspases-3, cytochrome c, HSP27, cIAP-1, cIAP-2, p53, and XIAP. The combinatorial drug showed promising anti-tumor efficacy in GBM xenograft model by reducing the tumor volume, suggesting it as an alternative drug to TMZ. Our findings indicate the coordinated administration of THTMP + T0 as an efficient therapy for inhibiting GBM cell proliferation.

Down regulation of lactate dehydrogenase initiates apoptosis in HeLa and MCF-7 cancer cells through increased voltage-dependent anion channel protein and inhibition of BCL2.

Malignant cells commonly use aerobic glycolysis for ATP production; this is known as the Warburg effect, where pyruvate is converted to lactate, by enzyme lactate dehydrogenase A (LDH-A). In this study, we have investigated the effect of inhibition of LDH-A on cells viability and identifying the mechanism of cell death in HeLa and MCF-7 cancer cells.Human cervical cancer HeLa cell line and breast cancer MCF-7 cell line were used to investigate the effect of inhibition of LDH-A by sodium oxamate on cell survival and proliferation using western blot, spectrophotometry, and immunofluorescent study.There was significant reduction in LDH-A (P < 0.001) and cell viability (P < 0.001) in a dose-dependent mode in both HeLa and MCF-7 SO-treated cancer cells. The voltage-dependent anion channel (VDAC) protein was significantly increased (P < 0.001) in association with decreased LDH-A. The proapoptotic proteins; cytochrome C (P < 0.001), BAX (P < 0.001), cleaved caspase-3 (P < 0.001), cleaved caspase-8 (P < 0.001), and cleaved caspase-9 (P < 0.001) were significantly increased in association with decreased LDH-A. While, the anti-apoptotic protein Bcl2 was significantly decreased (P < 0.001) in association with decreased LDH-A.We conclude that Inhibition of LDH-A can decrease cells viability through activation of intrinsic apoptotic pathway via increased VDAC protein and inhibition of Bcl2 as well as activation of the extrinsic apoptotic pathway through activation of caspase-8.

Subcellular Fractionation to Demonstrate Activation of Intrinsic Apoptotic Pathway.

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.

Cinnamaldehyde and Doxorubicin Co-Loaded Graphene Oxide Wrapped Mesoporous Silica Nanoparticles for Enhanced MCF-7 Cell Apoptosis.

BackgroundCombined chemotherapy is often affected by the different physicochemical properties of chemotherapeutic drugs, which should be improved by the reasonable design of co-loaded preparations.PurposeA kind of simple but practical graphene oxide (GO) wrapped mesoporous silica nanoparticles (MSN) modified with hyaluronic acid (MSN@GO-HA) were developed for the co-delivery of cinnamaldehyde (CA) and doxorubicin (DOX), in order to enhance their combined treatment on tumor cells and reduce their application defects.MethodsThe MSNCA@GODOX-HA was constructed by MSNCA (loading CA via physical diffusion) and GODOX-HA (modified with HA and loading DOX via π–π stacking) through the electrostatic adsorption, followed by the physicochemical characterization, serum stability and in vitro release study. Cytotoxicity on different cells was detected, followed by the tumor cell uptake tests. The intracellular reactive oxygen species (ROS) changes, mitochondrial functions and activities of caspase-3/-9 in MCF-7 cells were also evaluated, respectively.ResultsThe MSNCA@GODOX-HA nanoparticles kept stable in FBS solution and achieved pH-responsive release behavior, which was beneficial to increase the accumulation of CA and DOX in tumor cells to enhance the treatment. MSNCA@GODOX-HA exerted higher cytotoxicity to MCF-7 human breast cancer cells than H9c2 cardiac myocyte cells, which were not only attributed to the active targeting to tumor cells by HA, but also related with the activation of intrinsic apoptotic pathway in MCF-7 cells induced by CA, which was mediated by the specific ROS signal amplification and the interference with mitochondrial function. Moreover, the efficacy of DOX was also enhanced by the above process.ConclusionThe establishment of the MSNCA@GODOX-HA nanoparticles played a role in promoting strengths and restricting shortcomings of CA and DOX, thereby exerting their function and achieving efficient treatment against cancer.

Study on the relationships between molecular weights of chondroitin sulfate oligosaccharides and Aβ-induced oxidative stress and the related mechanisms.

In the present study, we studied anti-Alzheimer's disease (AD) activities of chondroitin sulfate (CS) oligosaccharides with different molecular weights. CS from shark cartilage was degraded by a recombinant CS endolyase, chondroitinase ABC I (CHSase ABC I), and CS disaccharide (DP2), tetrasaccharide (DP4), hexasaccharide (DP6), octasaccharide (DP8), decasaccharide (DP10) and dodecasaccharide (DP12) were obtained by separation with gel filtration. Anti-AD activities of CS oligosaccharides were assessed using Aβ-injured SH-SY5Y cells and BV2 cells. It was shown that CS oligosaccharides could block Aβ-induced oxidative stress, mitochondrial dysfunction and activation of intrinsic apoptotic pathway for SH-SY5Y cells. Furthermore, these activities increased with the increase of molecular weights. For Aβ-injured BV2 cells, CS oligosaccharides inhibited oxidative stress, the production of proinflammatory cytokines and the activation of toll-like receptor pathway, and CS DP2 had the best activity among them. In conclusion, CS oligosaccharides suppressed Aβ-induced oxidative stress and relevant injury in vitro, and these effects had different relationships with the molecular weights of CS oligosaccharides for different cell lines, which might be caused by different mechanisms.

A novel coordination complex of platinum (PT) induces cell death in colorectal cancer by altering redox balance and modulating MAPK pathway

BackgroundColorectal cancer (CRC) is a heterogeneous tumor having various genetic alterations. The current treatment options had limited impact on disease free survival due to therapeutic resistance. Novel anticancer agents are needed to treat CRC specifically metastatic colorectal cancer. A novel coordination complex of platinum, (salicylaldiminato)Pt(II) complex with dimethylpropylene linkage (PT) exhibited potential anti-cancer activity. In this study, we explored the molecular mechanism of PT-induced cell death in colorectal cancer.MethodsColony formation was evaluated using the clonogenic assay. Apoptosis, cell cycle analysis, reactive oxygen species, mitochondrial membrane potential and caspase-3/− 7 were assessed by flow cytometry. Glutathione level was detected by colorimetric assay. PT-induced alteration in pro-apoptotic/ anti-apoptotic proteins and other signaling pathways were investigated using western blotting. P38 downregulation was performed using siRNA.ResultsIn the present study, we explored the molecular mechanism of PT-mediated inhibition of cell proliferation in colorectal cancer cells. PT significantly inhibited the colony formation in human colorectal cancer cell lines (HT-29, SW480 and SW620) by inducing apoptosis and necrosis. This platinum complex was shown to significantly increase the reactive oxygen species (ROS) generation, depletion of glutathione and reduced mitochondrial membrane potential in colorectal cancer cells. Exposure to PT resulted in the downregulation of anti-apoptotic proteins (Bcl2, BclxL, XIAP) and alteration in Cyclins expression. Furthermore, PT increased cytochrome c release into cytosol and enhanced PARP cleavage leading to activation of intrinsic apoptotic pathway. Moreover, pre-treatment with ROS scavenger N-acetylcysteine (NAC) attenuated apoptosis suggesting that PT-induced apoptosis was driven by oxidative stress. Additionally, we show that PT-induced apoptosis was mediated by activating p38 MAPK and inhibiting AKT pathways. This was demonstrated by using chemical inhibitor and siRNA against p38 kinase which blocked the cytochrome c release and apoptosis in colorectal cancer cells.ConclusionCollectively, our data demonstrates that the platinum complex (PT) exerts its anti-proliferative effect on CRC by ROS-mediated apoptosis and activating p38 MAPK pathway. Thus, our findings reveal a novel mechanism of action for PT on colorectal cancer cells and may have therapeutic implication.

Combining triptolide with ABT-199 is effective against acute myeloid leukemia through reciprocal regulation of Bcl-2 family proteins and activation of the intrinsic apoptotic pathway

Bcl-2 inhibitors display an effective activity in acute myeloid leukemia (AML), but its clinical efficacy as a monotherapy was limited in part owing to failure to target other antiapoptotic Bcl-2 family proteins, such as Mcl-1. In this context, the combination strategy may be a promising approach to overcome this barrier. Here, we report the preclinical efficacy of a novel strategy combining ABT-199 with triptolide (TPL), a natural product extracted from a traditional Chinese medicine, in AML. Combination treatment exhibited markedly increased cytotoxicity in leukemic cells irrespective of p53 status while largely sparing normal cells of the hematopoietic lineage. Moreover, co-administration of ABT-199 with TPL dramatically suppressed leukemia progression as well as prolonged animal survival in a xenograft AML model. The potentiated effect of ABT-199 and TPL against AML was associated with activation of the mitochondrum-related intrinsic apoptotic pathway through a mechanism reciprocally modulating Bcl-2 family proteins. In this case, TPL not only downregulated Mcl-1 but also upregulated proapoptotic BH3-only proteins, thereby overcoming the resistance toward ABT-199. Conversely, ABT-199 abrogated Bcl-2-mediated cytoprotection against TPL. Together, these findings suggest that the regimen combining TPL and ABT-199 might be active against AML by inducing robust apoptosis through reciprocal regulation of anti- and proapoptotic Bcl-2 family proteins, therefore providing a strong rationale for the clinical investigation of this combination regimen for the treatment of AML.

Altered Mitochondrial Dynamics in Motor Neuron Disease: An Emerging Perspective.

Mitochondria plays privotal role in diverse pathways that regulate cellular function and survival, and have emerged as a prime focus in aging and age-associated motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Accumulating evidence suggests that many amyloidogenic proteins, including MND-associated RNA/DNA-binding proteins fused in sarcoma (FUS) and TAR DNA binding protein (TDP)-43, are strongly linked to mitochondrial dysfunction. Animal model and patient studies have highlighted changes in mitochondrial structure, plasticity, replication/copy number, mitochondrial DNA instability, and altered membrane potential in several subsets of MNDs, and these observations are consistent with the evidence of increased excitotoxicity, induction of reactive oxygen species, and activation of intrinsic apoptotic pathways. Studies in MND rodent models also indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting a causal role of mitochondrial dysfunction. Our recent studies, which demonstrated the involvement of specific defects in DNA break-ligation mediated by DNA ligase 3 (LIG3) in FUS-associated ALS, raised a key question of its potential implication in mitochondrial DNA transactions because LIG3 is essential for both mitochondrial DNA replication and repair. This question, as well as how wild-type and mutant MND-associated factors affect mitochondria, remain to be elucidated. These new investigation avenues into the mechanistic role of mitochondrial dysfunction in MNDs are critical to identify therapeutic targets to alleviate mitochondrial toxicity and its consequences. In this article, we critically review recent advances in our understanding of mitochondrial dysfunction in diverse subgroups of MNDs and discuss challenges and future directions.

Hsp90 inhibition aggravates adriamycin-induced podocyte injury through intrinsic apoptosis pathway

Podocyte injury leads to impaired filtration barrier function of the kidney that underlies the pathophysiology of idiopathic nephrotic syndrome (INS), the most common NS occurring in children. The heat shock protein 90 (Hsp90) is involved in the regulation of apoptosis in a variety of cell types, however, little is known about its role in podocytes and whether it associated with NS. Here, we show that Hsp90 is upregulated in glomeruli podocytes from mice with adriamycin (ADR)-induced nephropathy, and that it is also upregulated in an immortalized podocyte cell line treated with ADR in vitro, together suggesting an association of Hsp90 upregulation in podocytes with NS pathogenesis. Functionally, Hsp90 inhibition with PU-H71 aggravates ADR-induced podocyte apoptosis and worsens the impairment of filtration barrier function. Mechanistically, Hsp90 inhibition with PU-H71 enhances the activation of intrinsic apoptotic pathway, and moreover, blockade of podocyte apoptosis with zVAD-fmk (aVAD), a pan-caspase inhibitor, abrogates effects of Hsp90 inhibition on filtration barrier function of ADR-treated podocytes, thus demonstrating that Hsp90 inhibition aggravates ADR-induced podocyte injury through intrinsic apoptosis pathway. In sum, this study reveals a detrimental role of Hsp90 inhibition in podocyte injury, which may offer it as a potential therapeutic target in NS therapy.

Raptinal bypasses BAX, BAK, and BOK for mitochondrial outer membrane permeabilization and intrinsic apoptosis

Most antineoplastic chemotherapies eliminate cancer cells through activation of the mitochondria-controlled intrinsic apoptotic pathway. Therein, BAX, BAK, and/or BOK function as the essential pore-forming executioners of mitochondrial outer membrane permeabilization (MOMP). The activation threshold of BAX and BAK also correlates inversely with the required strength of an apoptotic stimulus to induce MOMP and thereby effectively determines a cell’s readiness to undergo apoptosis. Consequently, the ‘gatekeepers’ BAX and BAK emerged as therapeutic targets, but functional or genetic loss renders BAX/BAK-targeting strategies prone to fail. Here, we show that the small molecule Raptinal overcomes this limitation by triggering cytochrome c release in a BAX/BAK/BOK-independent manner. Raptinal exerts a dual cytotoxic effect on cancer cells by rapid activation of the intrinsic apoptotic pathway and simultaneous shutdown of mitochondrial function. Together with its efficacy to eliminate cancer cells in vivo, Raptinal could be useful in difficult-to-treat cancer entities harboring defects in the intrinsic apoptosis pathway.

Synthetic lethality of combined AT-101 with idarubicin in acute myeloid leukemia via blockade of DNA repair and activation of intrinsic apoptotic pathway.

Leukemia stem cells (LSCs) are deemed to the mainspring for treatment failure in acute myeloid leukemia (AML). Conventional chemotherapeutic drugs fail to eradicate leukemia stem cells, which becomes the root of drug resistance and disease recurrence. Hence, new therapeutic strategies targeting LSCs are supposed to be critical for patients with AML. Here we report that combination of Bcl-2 inhibitor AT-101 and chemotherapeutic drug idarubicin (IDA) results in synergistic lethality in CD34+CD38- leukemia stem-like cells sorted from KG-1α and Kasumi-1 AML cell lines and primary CD34+ AML cells in vitro while sparing the normal counterparts. In addition, combinatorial treatment also significantly inhibits the growth of patient-derived xenograft (PDX) mouse models generated from FLT3-ITDmut AML patient in vivo. Mechanistically, the synergistic effects of AT-101 with IDA to induce cell death are closely associated with blockage of DNA damage repair and thus activates the intrinsic apoptotic pathway. In summary, these findings suggest that combinatorial therapy with AT-101 and IDA selectively eliminates leukemia stem-like cells both in vitro and in vivo, representing a potent and alternative salvage therapy for the treatment of relapsed and refractory patients with AML.

Curcumin Induces Apoptotic Cell Death via Inhibition of PI3-Kinase/AKT Pathway in B-Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a significant cancer of children resulting from the clonal proliferation of lymphoid precursors with arrested maturation. Although chemotherapeutic approaches have been achieving successful remission for the majority of cases of childhood ALL, development of resistance to chemotherapy has been observed. Thus, new therapeutic approaches are required to improve patient's prognosis. Therefore, we investigated the anticancer potential of curcumin in ALL. We tested a panel of B-precursor ALL (B-Pre-ALL) cell lines with various translocations after treatment with different doses of curcumin. Curcumin suppresses the viability in a concentration-dependent manner in 697, REH, SupB15, and RS4;11 cells (doses from 0 to 80 μM). Curcumin induces apoptosis in B-Pre-ALL cell lines via activation of caspase-8 and truncation of BID. Curcumin treatment increased the ratio of Bax/Bcl-2 and resulted in a leaky mitochondrial membrane that led to the discharge of cytochrome c from the mitochondria to the cytoplasm, the activation of caspase 3 and the cleavage of PARP. Curcumin treatment of B-Pre-ALL cell lines induced a dephosphorylation of the constitutive phosphorylated AKT/PKB and a down-regulation of the expression of cIAP1, and XIAP. Moreover, curcumin mediates its anticancer activity by the generation of reactive oxygen species. Finally, the suboptimal doses of curcumin potentiated the anticancer activity of cisplatin. Altogether, these results suggest an important therapeutic role of curcumin, acting as a growth suppressor of B-Pre-ALL by apoptosis via inactivation of AKT/PKB and down-regulation of IAPs and activation of intrinsic apoptotic pathway via generation of Reactive Oxygen Species (ROS). Our interesting findings raise the possibility of considering curcumin as a potential therapeutic agent for the treatment of B-Pre-ALL.

Pendimethalin-induced oxidative stress, DNA damage and activation of anti-inflammatory and apoptotic markers in male rats

Male Wistar rats were exposed to herbicide, pendimethalin (PND) at varying oral doses of 62.5, 125 and 250 mg/kg b.w. for 14 days. Toxiological effects were assessed in terms of oxidative stress, DNA damage, histopathological alterations and induction of anti-inflammatory and apoptotic responses linked Bax, Bcl-2, IFN-γ, TNF-α and caspase-3 gene expression. In comparison with respective untreated controls, all exposure groups of PND exhibited significant changes in the oxidative stress markers (protein carbonylation and lipid peroxidation) and antioxidant defenses (GSH, SOD, CAT and GST) in liver and kidney tissues. The histopathological changes including leucocyte infiltration, pyknotic nuclei, necrosis, large bowman’s space, shrinked renal cortex, were observed in the liver and kidney tissues of PND exposed rats. Significant DNA damage was recorded through comet assay in liver and kidney cells of treated animals as compared to control. Alteration in anti-inflammatory and apoptotic genes expression determined by RT-PCR, revealed the activation of intrinsic apoptotic pathway(s) under the PND induced cellular stress. A pronounced increase in Bax expression, caspase-3 activities and decreased Bcl-2 expressions were also associated with PND-induced apoptosis. Data from this study suggests that PND induces cellular toxicity and genetic perturbations which can alter the normal cellular and physiological functioning in rats.

NeuroEPO Preserves Neurons from Glutamate-Induced Excitotoxicity.

Many experimental studies show that erythropoietin (EPO) has a neuroprotective action in the brain. EPO in acute and chronic neurological disorders, particularly in stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, has neuroprotective effects. We previously reported the neuroprotective effect of NeuroEPO, a low sialic form of EPO, against oxidative stress induced by glutamate excitotoxicity. In this paper, we analyze the effect of NeuroEPO against apoptosis induced by glutamate excitotoxicity in primary neuronal cultures obtained from the forebrains of Wistar rat embryos after 17 days of gestation. Excitotoxicity was induced after nine days of in vitro culture by treatment with a culture medium containing 100μM glutamate for 15 min. To withdraw glutamate, a new medium containing 100 ng NeuroEPO/mL was added. Apoptosis was analyzed after 24 h. Images obtained by phase contrast microscopy show that neurons treated with glutamate exhibit cell body shrinkage, loss of dendrites that do not make contact with neighboring cells, and that NeuroEPO was able to preserve the morphological characteristics of the control. Immunocytochemistry images show that the culture is essentially pure in neurons; that glutamate causes cell mortality, and that this is partially avoided when the culture medium is supplemented with NeuroEPO. Activation of intrinsic apoptotic pathways was analyzed. The decreases in Bcl-2/Bax ratio, increase in the release of cytochrome c, and in the expression and activity of caspase-3 observed in cells treated with glutamate, were restored by NeuroEPO. The results from this study show that NeuroEPO protects cortical neurons from glutamate-induced apoptosis via upregulation of Bcl-2 and inhibit glutamate-induced activation of caspase-3.

Lycosin-II Suppresses the Growth of Tumor Cells and Kills them Through Membrane Disruption and Apoptosis Induction

Recently, there has been a growing interest in the discovery of novel chemotherapeutic agents that have fewer side effects and are less likely to develop resistance. This study was conducted to examine the antitumor activity of lycosin-II, an antibacterial peptide isolated from the spider Lycosa singoriensis. Computational analysis revealed that lycosin-II has appropriate structural parameters (cationicity, α-helical conformation, amphipathicity) applicable for exhibiting antitumor activity. The results of MTT assay showed that lycosin-II possesses cytotoxicity (IC50 = 70.79 µg/ml) against the human colorectal cancer cell line HCT 116 as these cells were killed 30 min after treatment. Lycosin-II also exhibits a dose-dependent antiproliferative effect, as assessed using MTT assay coupled with direct cell counting as a measure of cell growth. Lactate dehydrogenase leakage assay and scanning electron microscopy revealed that lycosin-II kills tumor cells through cell membrane disruption. Apoptosis assays showed that Bax proapoptotic protein expression level and caspase-3 activity were increased after lycosin-II treatment, which indicates the activation of intrinsic apoptotic pathway. Altogether, these data indicate that lycosin-II suppresses the growth of tumor cells and kills them through membrane disruption and activation of apoptosis. However, lycosin-II exerts moderate hemolytic activity, which may be a major challenge in therapeutic applications. In conclusion, lycosin-II may serve as a template for designing novel antitumor peptides with low toxicity to normal cells for chemotherapeutic applications.

Selenotriapine – An isostere of the most studied thiosemicarbazone with pronounced pro-apoptotic activity, low toxicity and ability to challenge phenotype reprogramming of 3-D mammary adenocarcinoma tumors

Triapine, the most studied α-N-heterocyclic thiosemicarbazone, revealed potent activity against advanced leukemia, but was ineffective against a variety of solid tumors. Moreover, methemoglobinemia, which is a side effect of triapine administration, may limits all clinical application. To enhance anticancer activity and reduce side effects, we applied an isosteric replacement of sulfur to selenium atom was performed by synthesis and characterization of selenium triapine analog, 3-aminopyridine-2-carboxaldehyde selenosemicarbazone (selenotriapine). Compared to triapine, selenotriapine revealed superior pro-apoptotic activity with activation of intrinsic apoptotic pathway in both human monocytic leukemia (THP-1) and mammary adenocarcinoma (MCF-7) cell lines. For MCF-7 2-D cultures, selenotriapine induced notable increase in mitochondrial superoxide radical generation and dissipation of mitochondrial transmembrane potential. A significant delay in growth of MCF-7 spheroids (3-D culture) was accompanied by phenotypic stem cell reprogramming (Oct-4 expression). Additionally, selenotriapine demonstrated a very low toxicity profile as compared to triapine, confirmed over alleviated extent of methemoglobin formation and higher IC50 value in brine shrimp cytotoxicity assay.

Lipopolysaccharide Induces Human Pulmonary Micro-Vascular Endothelial Apoptosis via the YAP Signaling Pathway.

Gram-negative bacterial lipopolysaccharide (LPS) induces a pathologic increase in lung vascular leakage under septic conditions. LPS-induced human pulmonary micro-vascular endothelial cell (HPMEC) apoptosis launches and aggravates micro-vascular hyper-permeability and acute lung injury (ALI). Previous studies show that the activation of intrinsic apoptotic pathway is vital for LPS-induced EC apoptosis. Yes-associated protein (YAP) has been reported to positively regulate intrinsic apoptotic pathway in tumor cells apoptosis. However, the potential role of YAP protein in LPS-induced HPMEC apoptosis has not been determined. In this study, we found that LPS-induced activation and nuclear accumulation of YAP accelerated HPMECs apoptosis. LPS-induced YAP translocation from cytoplasm to nucleus by the increased phosphorylation on Y357 resulted in the interaction between YAP and transcription factor P73. Furthermore, inhibition of YAP by small interfering RNA (siRNA) not only suppressed the LPS-induced HPMEC apoptosis but also regulated P73-mediated up-regulation of BAX and down-regulation of BCL-2. Taken together, our results demonstrated that activation of the YAP/P73/(BAX and BCL-2)/caspase-3 signaling pathway played a critical role in LPS-induced HPMEC apoptosis. Inhibition of the YAP might be a potential therapeutic strategy for lung injury under sepsis.