Twenty-first century bioarchaeology: Taking stock and moving forward.

This article presents outcomes from a Workshop entitled "Bioarchaeology: Taking Stock and Moving Forward," which was held at Arizona State University (ASU) on March 6-8, 2020. Funded by the National Science Foundation (NSF), the School of Human Evolution and Social Change (ASU), and the Center for Bioarchaeological Research (CBR, ASU), the Workshop's overall goal was to explore reasons why research proposals submitted by bioarchaeologists, both graduate students and established scholars, fared disproportionately poorly within recent NSF Anthropology Program competitions and to offer advice for increasing success. Therefore, this Workshop comprised 43 international scholars and four advanced graduate students with a history of successful grant acquisition, primarily from the United States. Ultimately, we focused on two related aims: (1) best practices for improving research designs and training and (2) evaluating topics of contemporary significance that reverberate through history and beyond as promising trajectories for bioarchaeological research. Among the former were contextual grounding, research question/hypothesis generation, statistical procedures appropriate for small samples and mixed qualitative/quantitative data, the salience of Bayesian methods, and training program content. Topical foci included ethics, social inequality, identity (including intersectionality), climate change, migration, violence, epidemic disease, adaptability/plasticity, the osteological paradox, and the developmental origins of health and disease. Given the profound changes required globally to address decolonization in the 21st century, this concern also entered many formal and informal discussions.

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression.

BACKGROUND: Ceramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis. METHODS: The activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models. RESULTS: Exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model. CONCLUSION: We have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

Calcineurin-mediated dephosphorylation of eNOS at serine 116 affects eNOS enzymatic activity indirectly by facilitating c-Src binding and tyrosine 83 phosphorylation.

It has been shown previously that phosphorylation of the endothelial nitric oxide synthase (eNOS) at serine 116 (S116) under basal conditions suppresses eNOS enzymatic activity in endothelial cells. It has also been shown that vascular endothelial growth factor (VEGF) treatment of endothelial cells produces a rapid S116 dephosphorylation, which is blocked by the calcineurin inhibitor, cyclosporin A (CsA). In this study, we show that activation of eNOS in response to a variety of other eNOS-activating agonists and the cytosolic calcium-elevating agent, thapsigargin also involves CsA-inhibitable S116 dephosphorylation. Studies with the purified eNOS enzyme also demonstrate that neither mimicking phosphorylation at S116 nor phosphorylation of the purified enzyme at S116 in vitro has any effect on enzymatic activity. Phospho-mimicking, however, does interfere with the interaction of eNOS with c-Src, an interaction which is known to activate eNOS by phosphorylation at tyrosine 83 (Y83). Agonist-stimulated eNOS-Src complex formation, as well as agonist-stimulated Y83 phosphorylation, are blocked by calcineurin inhibition by CsA and by a cell-permeable calcineurin inhibitory peptide. Taken together, these data suggest a mechanism of eNOS regulation whereby calcineurin-mediated dephosphorylation of eNOS at S116 affects eNOS enzymatic activity indirectly, rather than directly, by facilitating c-Src binding and Y83 phosphorylation.

Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo.

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.

Cutting edge: IRF8 regulates Bax transcription in vivo in primary myeloid cells.

A prominent phenotype of IRF8 knockout (KO) mice is the uncontrolled expansion of immature myeloid cells. The molecular mechanism underlying this myeloproliferative syndrome is still elusive. In this study, we observed that Bax expression level is low in bone marrow preginitor cells and increases dramatically in primary myeloid cells in wt mice. In contrast, Bax expression level remained at a low level in primarymyeloid cells in IRF8 KO mice. However, in vitro IRF8 KO bone marrow-differentiated myeloid cells expressed Bax at a level as high as that in wild type myeloid cells. Furthermore, we demonstrated that IRF8 specifically binds to the Bax promoter region in primary myeloid cells. Functional analysis indicated that IRF8 deficiency results in increased resistance of the primary myeloid cells to Fas-mediated apoptosis. Our findings show that IRF8 directly regulates Bax transcription in vivo, but not in vitro during myeloid cell lineage differentiation.

Pin1 prolyl isomerase regulates endothelial nitric oxide synthase.

OBJECTIVE: The Pin1 prolyl isomerase acts in concert with proline-directed protein kinases to regulate function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. We sought to determine whether Pin1 interacts with endothelial nitric oxide synthase (eNOS) in endothelial cells in a manner that depends on proline-directed phosphorylation of the eNOS enzyme and whether this interaction influences basal or agonist-stimulated eNOS activity. METHODS AND RESULTS: Inhibitors of the extracellular-regulated kinase (ERK) 1/2 MAP kinases inhibit proline-directed phosphorylation of eNOS at serine 116 (Ser116) in bovine aortic endothelial cells (BAECs). Moreover, eNOS and Pin1 can be coimmunoprecipitated from BAECs only when Ser116 is phosphorylated. In addition, phosphomimetic Ser116Asp eNOS, but not wild-type eNOS, can be coimmunoprecipitated with Pin1 coexpressed in COS-7 cells. Inhibition of Pin1 in BAECs by juglone or by dominant negative Pin1 increases basal and agonist-stimulated NO release from the cells, whereas overexpression of wild-type Pin1 in BAECs suppresses basal and agonist-stimulated NO production. Overexpression of wild-type Pin1 in intact aortae also reduces agonist-induced relaxation of aortic rings. CONCLUSIONS: Our results demonstrate a novel form of eNOS regulation in endothelial cells and blood vessels through Ser116 phosphorylation-dependent interaction of eNOS with Pin1.

Activation of endothelial nitric oxide synthase by the pro-apoptotic drug embelin: Striking discrepancy between nitric oxide-mediated cyclic GMP accumulation and L-citrulline formation.

The benzoquinone derivative embelin is a multifunctional drug that not only induces apoptosis by inhibiting XIAP, the X chromosome-linked inhibitor of apoptosis protein, but also blocks nuclear factor-kappaB signaling pathways, thereby leading to down-regulation of a variety of gene products involved in tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. Here, we report that embelin activates and modulates l-arginine/nitric oxide/cyclic GMP signaling in cultured endothelial cells. Embelin elicited a rapid increase of intracellular free Ca(2+), leading to activation of endothelial nitric oxide synthase (eNOS) and NO-induced cGMP accumulation. While the cGMP response was comparable to that caused by other Ca(2+)-mobilizing agents, the stimulatory effect of embelin on l-citrulline formation (approximately 4-fold) was substantially lower than that observed upon activation of eNOS with the Ca(2+) ionophore A23187 (approximately 18-fold), the receptor agonist ATP (approximately 16-fold) or the sarco-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin (approximately 14-fold). The apparent discrepancy between NO/cGMP and l-citrulline formation in embelin-treated cells was not due to enhanced metabolism and/or efflux of l-citrulline, increased NO bioavailability, inhibition of cGMP hydrolysis, sensitization of soluble guanylate cyclase (sGC) to NO, or enhanced formation of a sGC/eNOS complex. Our puzzling observations suggest that embelin improves coupling of endothelial NO synthesis to sGC activation through mobilization of an as yet unrecognized signaling pathway.