Doxorubicin induces cell death in breast cancer cells regardless of Survivin and XIAP expression levels

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen.     

Musashi2 modulates K562 leukemic cell proliferation and apoptosis involving the MAPK pathway

The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment.     

Bleomycin induces alveolar epithelial cell death through JNK-dependent activation of the mitochondrial death pathway

Exposure to bleomycin in rodents induces lung injury and fibrosis. Alveolar epithelial cell death has been hypothesized as an initiating mechanism underlying bleomycin-induced lung injury and fibrosis. In the present study we evaluated the contribution of mitochondrial and receptor-meditated death pathways in bleomycin-induced death of mouse alveolar epithelial cells (MLE-12 cells) and primary rat alveolar type II cells. Control MLE-12 cells and primary rat alveolar type II cells died after 48 h of exposure to bleomycin. Both MLE-12 cells and rat alveolar type II cells overexpressing Bcl-X(L) did not undergo cell death in response to bleomycin. Dominant negative Fas-associating protein with a death domain failed to prevent bleomycin-induced cell death in MLE-12 cells. Caspase-8 inhibitor CrmA did not prevent bleomycin-induced cell death in primary rat alveolar type II cells. Furthermore, fibroblast cells deficient in Bax and Bak, but not Bid, were resistant to bleomycin-induced cell death. To determine whether the stress kinase JNK was an upstream regulator of Bax activation, MLE-12 cells were exposed to bleomycin in the presence of an adenovirus encoding a dominant negative JNK. Bleomycin-induced Bax activation was prevented by the expression of a dominant negative JNK in MLE-12 cells. Dominant negative JNK prevented cell death in MLE-12 cells and in primary rat alveolar type II cells exposed to bleomycin. These data indicate that bleomycin induces cell death through a JNK-dependent mitochondrial death pathway in alveolar epithelial cells.     

E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.     

E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.     

Matrine inhibits proliferation and induces apoptosis via BID-mediated mitochondrial pathway in esophageal cancer cells

Matrine, as a member of Sophora family, is an alkaloid found in plants, and produces plethora pharmacological effects, including anti-cancer effects. However, the mechanism involved remains largely unknown. This study is conducted to investigate the anti-cancer mechanisms of matrine in human esophageal cancer in vitro and in vivo. In human esophageal cancer cell Eca-109, matrine significantly decreased the cell viability in a dose-dependent manner, and induced apoptosis as well as cell cycle arrest in G0/G1 phase by up-regulation of P53 and P21. The expression of several apoptosis-related proteins in cells and tumor tissues were evaluated by Western blot analysis. We found that matrine induced cell apoptosis by down-regulation of the ratio of BCL-2/BID and increasing activation of caspase-9. Further studies indicated that matrine induced apoptosis of Eca-109 was through the mitochondria-mediated internal pathway, but not by death receptor-mediated extrinsic apoptotic pathway, which was confirmed by the fact that Bid translocated from the nucleus to mitochondria during the process of the apoptosis induced by matrine. In vivo study found that matrine effectively inhibited the tumor formation of Eca-109 cells in nude mice. Our study suggests that matrine could serve as a potential novel agent from natural products to treat esophageal cancer.     

Retinoid-regulated gene expression in normal and leukemic myeloid cells

Physiological concentrations of retinoic acid can induce acute alterations in the expression of the enzyme tissue transglutaminase in cultured macrophages. The induction of this enzyme offers a probe to study the mechanism of retinoid action in both normal and leukemic cells.     

Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway

Rhein is an anthraquinone compound enriched in the rhizome of rhubarb, a traditional Chinese medicine herb showing anti-tumor promotion function. In this study, we first reported that rhein could induce apoptosis in human promyelocytic leukemia cells (HL-60), characterized by caspase activation, poly(ADP)ribose polymerase (PARP) cleavage, and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6h. Mechanistic analysis demonstrated that rhein induced the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondrion to cytosol, and cleavage of Bid protein. Rhein also induced generation of reactive oxygen species (ROS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. However, these actions seem not to be associated with the apoptosis induction because antioxidants including N-acetyl cysteine (NAC), Tiron, and catalase did not block rhein-induced apoptosis, although they could block the generation of ROS and the phosphorylation of JNK and p38 kinase. Our data demonstrate that rhein induces apoptosis in HL-60 cells via a ROS-independent mitochondrial death pathway.     

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip_L expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosis-resistant. Autophagy (cellular “self digestion”) has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (     

The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.     

Exposure to the anti-TNF-alpha drug thalidomide induces apoptotic cell death in human T leukemic cells.

TNF-alpha is a pro-inflammatory cytokine that plays a key role in disorders due to HIV-1 infection and replication such as Kaposi sarcoma, wasting, aphthous ulcerations and progression to AIDS. The controversial drug thalidomide is anti-inflammatory, anti-angiogenic and a selective inhibitor of TNF-alpha that is being studied as a treatment for HIV-1-related disorders, immune disorders and cancer. The cellular and molecular mechanism of thalidomide is unclear despite renewed clinical interest in the drug. Previous data from this laboratory indicate that thalidomide decreases cell growth and cell-cell interactions of human T leukemic cells. The specific aim of the present study is to determine whether thalidomide administration induces cell death via apoptosis. Low dose thalidomide treatment of human T leukemic cells exhibited rapid increases in caspase-3 activity, annexin V-FITC binding and DNA disintegration that is characteristic of apoptosis. These data indicate that low doses of thalidomide signal human T leukemic cells to die by apoptosis, which is a possible method of altering inflammatory cells and inflammatory activities.     

Curcumin induces small cell lung cancer NCI-H446 cell apoptosis via the reactive oxygen species-mediated mitochondrial pathway and not the cell death receptor pathway

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.