DNA damage response (DDR) is a highly conserved genome surveillance mechanism that preserves cell viability in the presence of chemotherapeutic drugs. Hence, small molecules that inhibit DDR are expected to enhance the anti-cancer effect of chemotherapy. Through a recent chemical library screen, we identified shikonin as an inhibitor that strongly suppressed DDR activated by various chemotherapeutic drugs in cancer cell lines derived from different origins. Mechanistically, shikonin inhibited the activation of ataxia telangiectasia mutated (ATM), and to a lesser degree ATM and RAD3-related (ATR), two master upstream regulators of the DDR signal, through inducing degradation of ATM and ATR-interacting protein (ATRIP), an obligate associating protein of ATR, respectively. As a result of DDR inhibition, shikonin enhanced the anti-cancer effect of chemotherapeutic drugs in both cell cultures and in mouse models. While degradation of ATRIP is proteasome dependent, that of ATM depends on caspase- and lysosome-, but not proteasome. Overexpression of ATM significantly mitigated DDR inhibition and cell death induced by shikonin and chemotherapeutic drugs. These novel findings reveal shikonin as a pan DDR inhibitor and identify ATM as a primary factor in determining the chemo sensitizing effect of shikonin. Our data may facilitate the development of shikonin and its derivatives as potential chemotherapy sensitizers through inducing ATM degradation.

Diabetic nephropathy (DN) has been recognized as a severe complication of diabetes mellitus and a dominant pathogeny of end-stage kidney disease, which causes serious health problems and great financial burden to human society worldwide. Conventional strategies, such as renin-angiotensin-aldosterone system blockade, blood glucose level control, and bodyweight reduction, may not achieve satisfactory outcomes in many clinical practices for DN management. Notably, due to the multi-target function, Chinese medicine possesses promising clinical benefits as primary or alternative therapies for DN treatment. Increasing studies have emphasized identifying bioactive compounds and molecular mechanisms of reno-protective effects of Chinese medicines. Signaling pathways involved in glucose/lipid metabolism regulation, antioxidation, anti-inflammation, anti-fibrosis, and podocyte protection have been identified as crucial mechanisms of action. Herein, we summarize the clinical efficacies of Chinese medicines and their bioactive components in treating and managing DN after reviewing the results demonstrated in clinical trials, systematic reviews, and meta-analyses, with a thorough discussion on the relative underlying mechanisms and molecular targets reported in animal and cellular experiments. We aim to provide comprehensive insights into the protective effects of Chinese medicines against DN.

Pancreatic cancer is one of the most aggressive cancers with poor prognosis and a low 5-year survival rate. The family of P21-activated kinases (PAKs) appears to modulate many signaling pathways that contribute to pancreatic carcinogenesis. In this work, we demonstrated that PAK1 is a critical regulator in pancreatic cancer cell growth. PAK1-targeted inhibition is therefore a new potential therapeutic strategy for pancreatic cancer. Our small molecule screening identified a relatively specific PAK1-targeted inhibitor, CP734. Pharmacological and biochemical studies indicated that CP734 targets residue V342 of PAK1 to inhibit its ATPase activity. Further in vitro and in vivo studies elucidated that CP734 suppresses pancreatic tumor growth through depleting PAK1 kinase activity and its downstream signaling pathways. Little toxicity of CP734 was observed in murine models. Combined with gemcitabine or 5-fluorouracil, CP734 also showed synergistic effects on the anti-proliferation of pancreatic cancer cells. All these favorable results indicated that CP734 is a new potential therapeutic candidate for pancreatic cancer.

DNA damage response (DDR) relies on swift and accurate signaling to rapidly identify DNA lesions and initiate repair. A critical DDR signaling and regulatory molecule is the posttranslational modification poly(ADP-ribose) (PAR). PAR is synthesized by a family of structurally and functionally diverse proteins called poly(ADP-ribose) polymerases (PARPs). Although PARPs share a conserved catalytic domain, unique regulatory domains of individual family members endow PARPs with unique properties and cellular functions. Family members PARP-1, PARP-2, and PARP-3 (DDR-PARPs) are catalytically activated in the presence of damaged DNA and act as damage sensors. Family members tankyrase-1 and closely related tankyrase-2 possess SAM and ankyrin repeat domains that regulate their diverse cellular functions. Recent studies have shown that the tankyrases share some overlapping functions with the DDR-PARPs, and even perform novel functions that help preserve genomic integrity. In this review, we briefly touch on DDR-PARP functions, and focus on the emerging roles of tankyrases in genome maintenance. Preservation of genomic integrity thus appears to be a common function of several PARP family members, depicting PAR as a multifaceted guardian of the genome.

Previous studies have shown that patients with Takotsubo syndrome (TS) have supranormal nitric oxide signaling, and post-mortem studies of TS heart samples revealed nitrosative stress. Therefore, we first showed in a female rat model that isoproterenol induces TS-like echocardiographic changes, evidence of nitrosative stress, and consequent activation of the energy-depleting enzyme poly(ADP-ribose) polymerase-1. We subsequently showed that pre-treatment with an inhibitor of poly(ADP-ribose) polymerase-1 ameliorated contractile abnormalities. These findings thus add to previous reports of aberrant β-adrenoceptor signaling (coupled with nitric oxide synthase activation) to elucidate mechanisms of impaired cardiac function in TS and point to potential methods of treatment.

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 μM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 μM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

BACKGROUND: Technological advances including high-throughput sequencing have identified numerous tumor-specific genetic changes in pediatric and adolescent cancers that can be exploited as targets for novel therapies.
METHODS: This review provides a detailed overview of recent advances in the application of target-specific therapies for childhood cancers, either as single agents or in combination with other therapies. The review summarizes preclinical evidence on which clinical trials are based, early phase clinical trial results, and the incorporation of predictive biomarkers into clinical practice, according to cancer type.
CONCLUSIONS: There is growing evidence that molecularly targeted therapies can valuably add to the arsenal available for treating childhood cancers, particularly when used in combination with other therapies. Nonetheless the introduction of molecularly targeted agents into practice remains challenging, due to the use of unselected populations in some clinical trials, inadequate methods to evaluate efficacy, and the need for improved preclinical models to both evaluate dosing and safety of combination therapies.
CONCLUSIONS: The increasing recognition of the heterogeneity of molecular causes of cancer favors the continued development of molecularly targeted agents, and their transfer to pediatric and adolescent populations.

Shiga toxin (Stx) causes fatal systemic complications. Stx induces apoptosis, but the mechanism of which is unclear. We report that Stx induced rapid reduction of short-lived anti-apoptotic proteins followed by activation of caspase 9 and the progression of apoptosis. Proteasome inhibitors prevented the reduction of anti-apoptotic proteins, and inhibited caspase activation and apoptosis, suggesting that the reduction of anti-apoptotic proteins is a prerequisite for Stx-induced apoptosis. A clinically approved proteasome inhibitor, bortezomib, prolonged the survival of mice challenged by Stx. These results imply that proteasome inhibition may be a novel approach to prevent the fatal effects of Stx.

Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sjögren\'s syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 μg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 μg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Δψm), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Δψm, and generation of ROS, could be observed with soluble epratuzumab in Daudi cells co-cultivated with human umbilical vein endothelial cells. These results suggest that the in vivo mechanism of non-ligand-blocking epratuzumab may, in part, involve the unmasking of CD22 to facilitate the trans-interaction of B cells with vascular endothelium.

TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials.