Punicalagin promotes the apoptosis in human cervical cancer (ME-180) cells through mitochondrial pathway and by inhibiting the NF-kB signaling pathway

Increasing attention of plant derived therapeutic agents against cancer, investigating the anti-proliferative efficiency of plant derived chemicals have achieved increasing momentum for the design of anticancer drug. Punicalagin, dietary phytochemical altered the various cell signal transduction pathways associated with cell apoptosis and proliferation. This investigation was intended to examine the efficiency of punicalagin lying on cell viability so as to examine the molecular based punicalagin mechanism stimulated apoptosis via exploring the expression of Bcl-2 family proteins, and caspases also the cell cycle regulatory proteins p53 and NF-κB signaling in human cervical cancer cells. We also analyzed the morphological characteristic changes through mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, TUNEL assay, AO/EtBr analysis in cervical cancer cells. Our findings demonstrated that punicalagin repressed the viability of cervical cancer cells in a dosereliant mode via stimulating mitochondrial mediated apoptosis. Moreover, our this study demonstrated that punicalagin blocked cervical cancer cell proliferation and stimulated cell apoptosis by suppressing NF-kappa B activity. Hence our study suggested that punicalagin exhibits opposing actions on NF-kappa B signaling networks to block cancer cell progression acts as a classical candidate for anticancer drug designing.     

Akt is upstream and MAPKs are downstream of NF-κB in paclitaxel-induced survival signaling events, which are down-regulated by curcumin contributing to their synergism

Paclitaxel is the most promising chemotherapeutic agent of plant origin despite its high cost and dose-limiting toxicity. Our earlier report has shown that cervical cancer cells can be sensitized by curcumin to paclitaxel-induced apoptosis through down-regulation of NF-κB and Akt. In the present study we have attempted to decipher the signaling pathways regulating the synergism of paclitaxel and curcumin. The study has clearly proved that Akt and NF-κB function successively in the sequence of paclitaxel induced signaling events where Akt is upstream of NF-κB. While inhibition of NF-κB led to complete inhibition of the synergism of paclitaxel and curcumin, inhibition of Akt brought about only partial reduction of the same, suggesting that, apart from Akt, there are other pathways induced by paclitaxel leading to NF-κB activation, which are also down-regulated by curcumin. Inactivation of NF-κB did not affect the activation of Akt and survivin, while that of Akt significantly inhibited NF-κB and completely inhibited up-regulation of survivin. Up-regulation of Cyclin-D1, Cox-2, XIAP and cIAP1 and phosphorylation of MAPKs, were completely inhibited on inactivation of NF-κB assigning a key regulatory role to NF-κB in the synergistic effect of paclitaxel and curcumin. While up-regulation of survivin by paclitaxel is regulated by Akt, independent of NF-κB, inactivation of neither Akt nor NF-κB produced any change in Bcl-2 level suggesting a distinct pathway for its action. As curcumin could effectively down-regulate all these survival signals induced by paclitaxel, we suggest it as a potent chemosensitizer to improve the therapeutic index of paclitaxel.     

Radiation induces apoptosis primarily through the intrinsic pathway in mammalian cells

Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in “initiator” caspases, “effector” caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both “initiator” and “effector” caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.     

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.     

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca²⁺ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.     

IAPs: a central element in the NF-κB activating signaling pathway

The function of IAP has long been limited to an inhibition of apoptosis through their capacity to bind some caspases. Since the expression of these proteins is altered in some tumor samples, IAPs are targets for anticancer therapy and many small molecules have been designed for their capacity to inhibit IAP-caspase interaction. Unexpectedly, these molecules appeared to significantly affect NF-κB activation. In this review, we will discuss the central role of cIAP1, cIAP2 and XIAP in the regulation of NF-κB activating signaling pathways.     

The effect of NF-��B pathway on proliferation and apoptosis of human umbilical vein endothelial cells induced by intermittent high glucose

Our previous study found that blocking nuclear factor (NF)-κB signaling could protect human umbilical vein endothelial cells (HUVECs) from apoptosis and proliferation inhibition due to high glucose (HG). Intermittent HG makes glucose toxicity more significant. In this study, we aimed to investigate the effect of NF-κB pathway on HUVECs induced by intermittent HG (a daily alternating 5.5 or 30.5 mmol/l glucose). A recombinant adenovirus containing a RNAi cassette targeting the NF-κB/p65 gene was produced, and its silencing effect on p65 gene was detected by Western blot analysis in HUVECs cultured with intermittent HG. The subsequent effect on proliferation of HUVECs in the indicated conditions was measured by the AlamarBlue assay. The Bcl-2 expression was also detected by Western blot. The results showed that the expression of p65 protein could be inhibited efficiently by the RNAi adenovirus. Intermittent HG also induced the translocation of NF-κB in HUVECs. Inhibition of NF-κB with the RNAi adenovirus could prevent the effects. At the 6th day after HUVECs were exposed to intermittent HG, the proliferation of HUVECs with Ad-1566 was significantly higher than that of HUVECs with Ad-DEST (P < 0.01). Knockdown of NF-κB/p65 up-regulated the Bcl-2 expression of HUVECs under intermittent HG conditions (P < 0.01). These findings concluded that the NF-κB/p65-targeting RNAi adenovirus is an important tool, which can efficiently inhibit the expression of p65 gene in HUVECs. Intermittent HG reduces HUVECs proliferation with a concomitant increase in apoptosis. Knockdown of NF-κB/p65 partly protected HUVECs from proliferation inhibition and may reduce apoptosis.     

Bone marrow mesenchymal stem cells-derived exosomal microRNA-16-5p restrains epithelial-mesenchymal transition in breast cancer cells via EPHA1/NF-κB signaling axis

ObjectiveThis study intends to conquer the mystery of microRNA-16-5p/erythropoietin-producing hepatocellular A1/nuclear factor-κB signaling (miR-16-5p/EPHA1/NF-κB signaling) in breast cancer.MethodsExpression of miR-16-5p, EPHA1 and NF-κB signaling-related proteins were detected. Gene overexpression or silencing was used to examine the biological roles of bone marrow mesenchymal stem cells (BMSCs)-derived exo-miR-16-5p in breast cancer. The effect of exo-miR-16-5p on tumorigenesis of breast cancer was confirmed by the xenograft nude mouse model.ResultsLow miR-16-5p and high EPHA1 expression were examined in breast cancer. BMSCs-derived exosomes, up-regulated miR-16-5p or down-regulated EPHA1 restrained epithelial-mesenchymal transition (EMT) of breast cancer cells and tumor growth in nude mice. Down-regulated miR-16-5p or up-regulated EPHA1 activated NF-κB signaling. Knockdown of EPHA1 or inhibition of NF-κB signaling reversed the effects of down-regulated miR-16-5p on breast cancer cells.ConclusionBMSCs-derived exosomal miR-16-5p hinders breast cancer cells progression via EPHA1/NF-κB signaling axis.     

Overcoming resistance of cancer cells to apoptosis

Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to the concept that development of cancers required the simultaneous acquisition, not only of deregulated cell division, but also of resistance to programmed cell death or apoptosis. Apoptosis is arguably the common pathway to cell death resulting from a range of therapeutic initiatives, so that understanding the basis for the resistance of cancer cells to apoptosis may hold the key to development of new treatment initiatives. Much has already been learnt about the apoptotic pathways in cancer cells and proteins regulating these pathways. In most cells, apoptosis is dependent on the mitochondrial dependent pathway. This pathway is regulated by pro- and anti-apoptotic members of the Bcl-2 family, and manipulation of these proteins offers scope for a number of treatment initiatives. Effector caspases activated by the mitochondrial pathway or from death receptor signaling are under the control of the inhibitor of apoptosis protein (IAP) family. Certain proteins from mitochondrial can, however, competitively inhibit their binding to effector caspases. Information about the structure of these proteins has led to initiatives to develop therapeutic agents to block the IAP family. In addition to development of selective agents based on these two (Bcl-2 and IAP) protein families, much has been learnt about signal pathways that may regulate their activity. These in turn might provide additional approaches based on selective regulators of the signal pathways.     

Carnosic acid modulates Akt/IKK/NF-κB signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells

This study investigates the efficacy of carnosic acid (CA), a polyphenolic diterpene, isolated from the plant rosemary (Rosemarinus officinalis), on androgen-independent human prostate cancer PC-3 cells. CA induced anti-proliferative effects in PC-3 cells in a concentration- and time-dependent manner, which was due to apoptotic induction as evident from flow-cytometry, DNA laddering and TUNEL assay. Apoptosis was associated with the activation of caspase-8, -9, -3 and -7, increase in Bax:Bcl-2 ratio, release of cytochrome-c and decrease in expression of inhibitor of apoptosis (IAP) family of proteins. Apoptosis was attenuated upon pretreatment with specific inhibitors of caspase-8 (Z-IETD-fmk) and caspase-9 (Z-LEHD-fmk) suggesting the involvement of both intrinsic and extrinsic apoptotic cascades. Further, apoptosis resulted from the inhibition of IKK/NF-κB pathway as evident from decreased DNA binding activity, nuclear translocation of p50 and p65 and IκBα phosphorylation. The down-regulation of IKK/NF-κB was associated with inhibition of Akt phosphorylation and its kinase activity with a concomitant increase in the serine/threonine protein phosphatase 2A (PP2A) activity. Pharmacologic inhibition of PP2A by okadaic acid and calyculin A, significantly reversed CA-mediated apoptotic events in PC-3 cells indicating that CA induced apoptosis by activation of PP2A through modulation of Akt/IKK/NF-κB pathway. In addition, CA induced apoptosis in another androgen refractory prostate cancer DU145 cells via intrinsic pathway as evidenced from the activation of caspase 3, cleavage of PARP, increase in Bax:Bcl-2 ratio and cytochrome-c release. Carnosic acid, therefore, may have the potential for use in the prevention and/or treatment of prostate cancer.     

Inactivation of Bcl-2 through IκB kinase (IKK)-dependent phosphorylation mediates apoptosis upon exposure to 4-hydroxynonenal (HNE)

Apoptosis of macrophage foam cells loaded with modified/oxidized lipids is implicated in destabilization of advanced atherosclerotic plaques in humans. Concentration of HNE, main aldehydic product of plasma LDL peroxidation, elevates in atherosclerotic lesions as well as in cultured cells under oxidative stress. Although this reactive aldehyde has been shown to promote apoptosis with the involvement of p38 MAPK and JNK in various mammalian cell lines, roles of B-cell lymphoma 2 (Bcl-2) family proteins remain to be deciphered. We demonstrated that HNE-induced apoptosis was accompanied by concurrent downregulations of antiapoptotic Bcl-x(L) and Mcl-1 as well as upregulation of proapoptotic Bak. Furthermore, phoshorylation of Bcl-2 at Thr56, Ser70, and probably more phosphorylation sites located on N-terminal loop domain associated with HNE-induced apoptosis in both U937 and HeLa cells while ectopic expression of a phospho-defective Bcl-2 mutant significantly attenuated apoptosis. In parallel to this, HNE treatment caused release of proapoptotic Bax from Bcl-2. Pharmacological inhbition of IKK inhibited HNE-induced Bcl-2 phosphorylation. Similarly, silencing IKKα and -β both ended up with abrogation of Bcl-2 phosphorylation along with attenuation of apoptosis. Moreover, both IKKα and -β coimmunoprecipitated with Bcl-2 and in vitro kinase assay proved the ability of IKK to phosphorylate Bcl-2. In view of these findings and considering HNE inhibits DNA-binding activity of nuclear factor-κB (NF-κB) through prevention of IκB phosphorylation/ubiquitination/proteolysis, IKK appears to directly interfere with Bcl-2 activity through phosphorylation in HNE-mediated apoptosis independent of NF-κB signaling.     

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Clinical case report of patients with osteosarcoma and anticancer benefit of calycosin against human osteosarcoma cells

Osteosarcoma (OS) is a malignant neoplasia in bone, characterized with main occurrence in teenagers. Calycosin (CC), a bioactive compound, is found to play potent pharmacological effects against cancer. Our previous study indicates CC-exerted benefits for anti-OS effect. However, further molecular mechanism behind this action needs to be investigated. In this study, human OS samples and clinical data were collected and used for further test and analysis. In addition, human osteosarcoma cell line (143B) and tumor-xenograft nude mice were used to evaluate antineoplastic activities of CC through a series of biochemical methods and immunoassays, respectively. Compared with non-OS controls, human OS samples showed increased levels of neoplastic microRNA-223 (miR-223), and elevated expressions of NF-κBp65, IκBα proteins in tumor cells. In cell culture study, CC-treated 143B cells showed reduced cell growth, increased lactic dehydrogenase (LD) content, and downregulated cellular miR-223 level. Immunolabeled cells of proliferating cell nuclear antigen, B-cell lymphoma 2 (Bcl-2), poly(ADP-ribose) polymerase (PARP) in CC treatments were decreased dose-dependently, while caspase-3 positive cells were elevated. Further, protein expressions of NF-κBp65, IκBα in CC-treated cells were downregulated. In addition, tumor-xenograft nude mice followed by CC treatments exhibited reductions of tumor mass, miR-223 levels, and Bcl-2, PARP-positive cells, as well as downregulations of NF-κBp65, IκBα protein expressions in OS samples. Taken together, these experimental findings reveal that CC exhibits potential pharmacological activities against OS through inducing apoptosis and inhibiting miR-223-IκBα signaling pathway in neoplastic cells.     

Characterization of antioxidant and anti-inflammatory activities of bioactive fractions recovered from a glucose?lysine Maillard reaction model system

A glucose-lysine (Glu-Lys) Maillard reaction mixture heated at 121°C for 60 min was processed by ultrafiltration, ethyl acetate extraction, and semi-preparative HPLC to recover a bioactive fraction, termed F3. F3, characterized by spectral analysis to contain three distinct components, inhibited NO and IL-8 by 70 and 61%, respectively, at a concentration of 50 μg/ml in inflamed Caco-2 cells induced by IFN-γ and phorbol 12-myristate 13-acetate (PMA). F3 significantly (P < 0.05) down-regulated several genes involved in nuclear factor kappa B (NF-κB) signaling pathway. These genes included the cytokine receptors, TNFRSF10A and TNFRSF10B; receptor-associated proteins, IRAK2 and TICAM1; the inhibitor κB kinase, IKBKE; the NF-κB inhibitor, NFKBIA; and the NF-κB subunits, REL, RELA, and RELB. F3 also down-regulated the NF-κB responsive genes IL-8, NOS2, and ICAM1, attenuated the gene expression of peroxidases such as DUOX1 and DUOX2, and relieved the down-regulated GCFHR that are involved in the biosynthesis of NO and TROAP, a gene suppressed by NO. The anti-inflammatory activity of F3 was mediated through multiple processes that included regulation of gene expressions involved in NF-κB signaling, the inhibition of IL-8 and iNOS translation, a decrease in NO synthesis and attenuating oxidative stress in inflamed Caco-2 cells. Our results show that MRP components have the potential to suppress inflammation in IFN-γ and PMA-induced Caco-2 cells.     

Pomegranate reverses methotrexate-induced oxidative stress and apoptosis in hepatocytes by modulating Nrf2-NF-κB pathways

The clinical efficacy of the widely used chemotherapeutic drug methotrexate (MTX) is limited due to its associated hepatotoxicity. Pomegranate polyphenols are of huge health benefits and known to possess remarkable antioxidant properties capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. In this study, we explored the protective role of pomegranate fruit extract (PFE) in ameliorating MTX-induced hepatic damage. Male Swiss albino mice exposed to MTX (20 mg/kg body weight) exhibited distinct markers of toxicity such as increased activities of enzymes alanine transaminase, aspartate transaminase, lactate dehydrogenase and alkaline phosphatase and also increased oxidative stress in liver evidenced by increased ROS generation and lipid peroxidation. Decrease in reduced glutathione levels, superoxide dismutase, catalase, hepatic heme oxygenase 1 and NQO-1 activities were also observed. Tracing the signal transduction pathways, it was seen that MTX exposure significantly increased nuclear translocation of NF-κB coupled with increase in phosphorylated Iκ-B and down-regulation of NF-kappaB-dependent antiapoptotic protein Bcl-2. Treatment with MTX increased the expression of the apoptotic enhancer Rho/Cdc42 as well as the phosphorylation of SAPK/JNK. A shift in the Bax/Bcl-2 ratio towards apoptosis and increase in the caspase 3 level was also evident. Administration of PFE for 7 consecutive days before and after MTX challenge suppressed MTX-induced cell death, mitigated the injurious effects of MTX and offered protection against apoptosis. PFE was shown to reduce ROS generation in hepatocytes by activating the Nrf2-ARE pathway and inhibiting NF-κB as a consequence of which the antioxidant defense mechanism in the liver was up-regulated, thereby conferring protection against MTX-induced hepatotoxicity and apoptosis.     

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.