Ciclopirox olamine (CPX), a fungicide, has been demonstrated as a potential anticancer agent. However, the underlying anticancer mechanism is not well understood. Here, we found that CPX induced autophagy in human rhabdomyosarcoma (Rh30 and RD) cells. It appeared that CPX-induced autophagy was attributed to induction of reactive oxygen species (ROS), as N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, prevented this process. Furthermore, we observed that CPX induced activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK, which was also blocked by NAC. However, only inhibition of JNK (with SP600125) or expression of dominant negative c-Jun partially prevented CPX-induced autophagy, indicating that ROS-mediated activation of JNK signaling pathway contributed to CPX-induced autophagy. Of interest, inhibition of autophagy by chloroquine (CQ) enhanced CPX-induced cell death, indicating that CPX-induced autophagy plays a pro-survival role in human rhabdomyosarcoma cells. Our finding suggests that the combination with autophagy inhibitors may be a novel strategy in potentiating the anticancer activity of CPX for treatment of rhabdomyosarcoma.

Dihydroartemisinin (DHA), an antimalarial drug, has previously unrecognized anticancer activity, and is in clinical trials as a new anticancer agent for skin, lung, colon and breast cancer treatment. However, the anticancer mechanism is not well understood. Here, we show that DHA inhibited proliferation and induced apoptosis in rhabdomyosarcoma (Rh30 and RD) cells, and concurrently inhibited the signaling pathways mediated by the mammalian target of rapamycin (mTOR), a central controller for cell proliferation and survival, at concentrations (<3 μM) that are pharmacologically achievable. Of interest, in contrast to the effects of conventional mTOR inhibitors (rapalogs), DHA potently inhibited mTORC1-mediated phosphorylation of p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1 but did not obviously affect mTORC2-mediated phosphorylation of Akt. The results suggest that DHA may represent a novel class of mTORC1 inhibitor and may execute its anticancer activity primarily by blocking mTORC1-mediated signaling pathways in the tumor cells.

Perturbations to endoplasmic reticulum (ER) morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that a human cytomegalovirus (HCMV) gene product, UL148, profoundly reorganizes the ER during infection and is sufficient to do so when expressed on its own. Our results reveal that UL148-dependent reorganization of the ER is a prominent feature of HCMV-infected cells. Moreover, we find that this example of virally induced organelle remodeling requires the integrated stress response (ISR), a stress adaptation pathway that contributes to a number of disease states. Since ER reorganization accompanies roles of UL148 in modulation of HCMV cell tropism and in evasion of antiviral immune responses, our results may have implications for understanding the mechanisms involved. Furthermore, our findings provide a basis to utilize UL148 as a tool to investigate organelle responses to stress and to identify novel drugs targeting the ISR.

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.

24 INTRODUCTION. 61UL148 is a human cytomegalovirus (HCMV) ER-resident glycoprotein that plays 62 roles in evasion of cell-mediated immunity and shows intriguing effects on cell tropism. 63During infection of epithelial cells, viruses disrupted for UL148 replicate to produce 64 roughly 100-fold enhanced levels of infectious progeny virions compared to wildtype (1). 65These effects correlate with reduced expression of glycoprotein O (gO), a subunit of a 66 heterotrimeric viral glycoprotein H (gH) / glycoprotein L (gL) complex (gH/gL/gO) on the 67 virion envelope that is required for the infectivity of cell-free virions (2-4), and which 68 endows the virus with the capacity to utilize the platelet derived growth factor receptor a 69 (PDGFRa) as an entry receptor (5-7). Accordingly, UL148 has been found to stabilize 70 immature forms of gO prior their assembly into gH/gL/gO heterotrimers (1, 8). Despite 71 that UL148 does not stably associate with gO, the data suggest that it may interact with 72 gH (1). 73UL148 also physically associates with CD58 (LFA-3), a co-stimulatory ligand for 74 natural killer cells and T-lymphocytes, preventing its presentation at cell surface (9). 75Although the mechanisms by which UL148 stabilizes gO and retains CD58 within the 76 ER remain unknown, UL148 strongly contributes to activation of the unfolded protein 77 response (UPR) during infection, and is sufficient to activate the UPR when ectopically 78 expressed in non-infected cells (10). UL148 co-purifies from infected cells with SEL1L, 79 an adaptor subunit of ER-based E3 ubiquitin ligase Hrd1 that plays important roles in 80 ER-associated degradation (ERAD) of terminally misfolded glycoproteins (8). This 81 suggests a physical interaction with ERAD machinery, which may be germane to the 82 mechanism by which UL148 activates the UPR. 83 93 6 RESULTS. 94 95 UL148 causes reorganization of ER quality control proteins into unusual globular 96 structures. 97The HCMV ER-resident glycoprotein UL148 was previously observed to co-98 localize with the ER marker calnexin during infection (1). Nonetheless, calnexin staining 99 did not show the uniform reticular pattern characteristic for the ER marker. We later 100 noticed that cells infected with a UL148-null virus showed uniform calnexin staining (see 101 below). To formally determine whether UL148 influences calnexin localization, we 102 compared fibroblasts at four days post-infection with either wildtype (TB_WT) or UL148-103 null mutant (TB_148STOP) viruses derived from an infectious bacterial artificial 104 chromosome (BAC) clone of HCMV strain TB40/E (FIG 1). In cells infected with 105 wildtype virus, calnexin antibodies stained unusual globular structures at the cell 106 periphery, as expected (1) (FIG 1A, 1C, SI FIG S1A). However, in cells infected with 107 the UL148-null virus, calnexin staining was uniform throughout the cytosol (FIG 1B-C), 108 as would be expected for an ER marker in uninfected cells. The staining pattern for 109 Hrd1, another ER marker, likewise indicated accumula...