Supplementary Material from Mitogen-activated protein kinase kinase 1/2 inhibitors and 17-allylamino-17-demethoxygeldanamycin synergize to kill human gastrointestinal tumor cells <i>in vitro</i> via suppression of c-FLIP-s levels and activation of CD95
Abstract Infection with the parasitic nematode Nippostrongylus brasiliensis induces a potent Th2 response; however, little is known about early stages of the innate response that may contribute to protective immunity. To examine early events in this response, chemokine expression in the draining lymph node was examined after N. brasiliensis inoculation. Pronounced increases of several chemokines, including CCL2, were observed. Compared with wild-type mice, elevations in a Gr-1bright population in the draining lymph node was significantly decreased in CCL2−/− mice after N. brasiliensis inoculation. Further flow cytometric and immunofluorescent analysis showed that in wild-type mice, Gr-1+ cells transiently entered and exited the draining lymph node shortly after N. brasiliensis inoculation. The Gr-1bright population was comprised of neutrophils expressing TGF-β and TNF-α. Following Gr-1+ cell depletion, N. brasiliensis infection resulted in transient, but significantly increased levels of IFN-γ, increased serum IgG2a, reduced Th2 cytokines and serum IgE, greatly increased mortality, and delayed worm expulsion. Furthermore, bacteria were readily detected in vital organs. Infection of Gr-1+ cell-depleted mice with N. brasiliensis larvae that were pretreated with antibiotics prevented bacterial dissemination, Th1 inflammatory responses, and decreases in host survival. This study indicates that parasitic nematodes can be an important vector of potentially harmful bacteria, which is typically controlled by CCL2-dependent neutrophils that ensure the optimal development of Th2 immune responses and parasite resistance.
Components of the type 2 immune response may mediate host protection against both helminthic parasites and harmful allergic responses. A central player in this response is the T-helper 2 (Th2) effector cell, which produces interleukin (IL)-4, IL-5, IL-13, and other Th2 cytokines during the primary and memory response. Specific aspects of the parasite that trigger Th2-cell differentiation are not yet defined. Furthermore, the cell types and cell surface and secreted molecules that provide the immune milieu required for the development of Th2 effector cells and also Th2 memory cells are not well understood. They will probably vary with the particular helminth or other antigen inducing the Th2 response. We have used third stage larvae of intestinal nematode parasites as adjuvants to promote naïve nonparasite antigen-specific T cells to differentiate into Th2 cells. This model system avoids possible parasite antigen-specific T-cell clones or cross-reactive memory T cells that may preferentially differentiate into Th2 effector cells during the course of infection and confound the stereotypical components of parasite-induced Th2 cell differentiation. We have found that these parasites have a potent adjuvant effect and have used our model system to begin to investigate the events that lead to the development of polarized Th2 cells in vivo.
Prior studies in breast cancer cells have shown that lapatinib and obatoclax interact in a greater than additive fashion to cause cell death and do so through a toxic form of autophagy. The present studies sought to extend our analyses to the central nervous system (CNS) tumor cells and to further define mechanisms of drug action. Lapatinib and obatoclax killed multiple CNS tumor isolates. Cells lacking PTEN (phosphatase and tensin homolog on chromosome 10) function were relatively resistant to drug combination lethality; expression of PTEN in PTEN-null cells restored drug sensitivity, and knockdown of PTEN promoted drug resistance. On the basis of knockdown of ERBB1-4 (erythroblastic leukemia viral oncogene homolog 1–4), we discovered that the inhibition of ERBB1/3/4 receptors were most important for enhancing obatoclax lethality rather than ERBB2. In parallel, we noted in CNS tumor cells that knockdown of BCL-XL (B-cell lymphomaextra large)and MCL-1 (myeloid cell leukemia-1) interacted in an additive fashion to facilitate lapatinib lethality. Pretreatment of tumor cells with obatoclax enhanced the lethality of lapatinib to a greater extent than concomitant treatment. Treatment of animals carrying orthotopic CNS tumor isolates with lapatiniband obatoclax-prolonged survival. Altogether, our data show that lapatinib and obatoclax therapy could be of use in the treatment of tumors located in the CNS.
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.
The ability of human chorionic gonadotropin (hCG) to modify prostate carcinoma viability in vitro and in vivo when combined with the HMG CoA reductase inhibitor lovastatin and ionizing radiation was investigated. Treatment of PC-3 cells in vitro with hCG caused a modest increase in numbers of non-viable cells within 96h. Treatment of cells with hCG followed by exposure to the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The cytotoxic effects of hCG were blocked by expression of BCL-XL and dominant negative caspase 9. Treatment of mice bearing PC-3 flank tumors with lovastatin and hCG significantly reduced tumor volume within 7 days; this was also reflected in decreased ex vivo colony survival of the cells which correlated with increased cleavage of pro-caspase 3 and reduced Ki67 immuno-reactivity. In vitro, treatment of PC-3 cells with hCG followed by exposure to ionizing radiation enhanced the cytotoxic effects of hCG, that was further enhanced by lovastatin. In vivo, hCG radiosensitized PC-3 tumors and significantly enhanced the lethality of hCG and lovastatin treatment. Collectively, our findings argue that treatment of PC-3 prostate cancer tumors with hCG, lovastatin and radiation represents a potential novel therapeutic approach.
