Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2‐mediated nitric oxide synthase signaling

Cold atmospheric plasma (CAP) represents a promising therapy for selectively cancer killing. However, the mechanism of CAP‐induced cancer cell death remains unclear. Here, we identified the tumor necrosis factor‐family members, especially Fas, and overloaded intracellular nitric oxide participated in CAP induced apoptosis in A375 and A875 melanoma cell lines, which was known as extrinsic apoptosis pathway. This progress was mediated by antagonistic protein of reactive oxygen species, Sestrin2. The over expression of Sestrin2 induced by plasma treatment resulted in phosphorylation of p38 mitogen‐activated protein kinase (MAPK), followed by increased expression of nitric oxide synthase (iNOS), Fas and Fas ligand. Depletion of Sestrin2 reduced iNOS and Fas expression, which was associated with reduction of plasma‐induced apoptosis. In contrast, inhibition of iNOS activity and phosphorylation of p38 did not alter Sestrin2 expression in plasma‐treated melanoma cells. Taken together, cold atmospheric plasma increases Sestrin2 expression and further activates downstream iNOS, Fas and p38 MAPK signaling to induce apoptosis of melanoma cell lines. These findings suggest a previously unrecognized mechanism in melanoma cells response to cold atmospheric plasma therapy.      

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

Activation of Tyk2 and Stat3 is required for the apoptotic actions of interferon-beta in primary pro-B cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.     

Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of Fas

Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.     

Gene transfer of tissue inhibitor of metalloproteinases-3 reverses the inhibitory effects of TNF-alpha on Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts

Apart from counteracting matrix metalloproteinases, tissue inhibitor of metalloproteinases-3 (TIMP-3) has proapoptotic properties. These features have been attributed to the inhibition of metalloproteinases involved in the shedding of cell surface receptors such as the TNFR. However, little is known about effects of TIMP-3 in cells that are not susceptible to apoptosis by TNF-alpha. In this study, we report that gene transfer of TIMP-3 into human rheumatoid arthritis synovial fibroblasts and MRC-5 human fetal lung fibroblasts facilitates apoptosis and completely reverses the apoptosis-inhibiting effects of TNF-alpha. Although TNF-alpha inhibits Fas/CD95-induced apoptosis in untransfected and mock-transfected cells, fibroblasts ectopically expressing TIMP-3 are sensitized most strongly to Fas/CD95-mediated cell death by TNF-alpha. Neither synthetic MMP inhibitors nor glycosylated bioactive TIMP-3 are able to achieve these effects. Gene transfer of TIMP-3 inhibits the TNF-alpha-induced activation of NF-kappaB in rheumatoid arthritis synovial fibroblasts and reduces the up-regulation of soluble Fas/CD95 by TNF-alpha, but has no effects on the cell surface expression of Fas. Collectively, our data demonstrate that intracellularly produced TIMP-3 not only induces apoptosis, but also modulates the apoptosis-inhibiting effects of TNF-alpha in human rheumatoid arthritis synovial fibroblast-like cells. Thus, our findings may stimulate further studies on the therapeutic potential of gene transfer strategies with TIMP-3.     

Immunosensitization of melanoma tumor cells to non-MHC Fas-mediated killing by MART-1-specific CTL cultures

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.