Modified dendritic cell-derived exosomes activate both NK cells and T cells through the NKG2D/NKG2D-L pathway to kill CML cells with or without T315I mutation.

BACKGROUND: Tyrosine kinase inhibitors have achieved quite spectacular advances in the treatment of chronic myeloid leukemia (CML), but disease progression and drug resistance that related to the T315I mutation, remain major obstacles. Dendritic cell-derived exosomes (Dex) induce NK cell immunity, but have yet to achieve satisfactory clinical efficacy. An approach to potentiate antitumor immunity by inducing both NK- and T-cell activation is urgently needed. Retinoic acid early inducible-1γ (RAE-1γ), a major ligand of natural killer group 2 member D (NKG2D), plays an important role in NK-cell and T-lymphocyte responses. We generated RAE-1γ enriched CML-specific Dex (CML-RAE-1γ-Dex) from dendritic cells (DCs) pulsed with lysates of RAE-1γ-expressing CML cells or T315I-mutant CML cells, aiming to simultaneously activate NK cells and T lymphocytes. METHODS: We generated novel CML-RAE-1γ-Dex vaccines, which expressed RAE-1γ, and were loaded with CML tumor cell lysates. NK cells or T lymphocytes were coincubated with CML-RAE-1γ-Dex vaccines. Flow cytometry was performed to evaluate the activation and proliferation of these immune cells. Cytokine production and cytotoxicity toward CML cells with or without the T315I mutation were detected by ELISPOT, ELISA and LDH assays. CML models induced by BCR-ABL or BCR-ABLT315I were used to determine the immunological function of Dex in vivo. RESULTS: Herein, CML-RAE-1γ-Dex were prepared. CML-RAE-1γ-Dex effectively enhanced the proliferation and effector functions of NK cells, CD4+ T cells and CD8+ T cells, which in turn produced strong anti-CML efficacy in vitro. Moreover, CML-RAE-1γ-Dex-based immunotherapy inhibited leukemogenesis and generated durable immunological memory in CML mouse models. Similar immune responses were also observed with imatinib-resistant CML cells carrying the T315I mutation. CONCLUSIONS: This approach based on CML-RAE-1γ-Dex vaccines may be a promising strategy for CML treatment, especially for cases with the T315I mutation.

Mebendazole exhibits potent anti-leukemia activity on acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most common types of acute leukemia in adults with the lowest survival rate of all leukemia. Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure. Thus, finding new drugs with anti-leukemia activities and minimal side effect is urgently needed. Here through screening more than 1000 drugs approved by the Food and Drug Administration (FDA) of United States, the anthelmintic drug mebendazole (MBZ) was found to inhibit the growth of AML cell lines (THP-1, U937, NB4 and K562) and bone marrow mononuclear cells (BM-MNCs) from AML patients at pharmacologically achievable concentrations. In contrast, similar concentration of MBZ had little inhibitory effect on the growth of normal peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC). In addition, MBZ induced mitotic arrest and mitotic catastrophe in AML cells based on nuclear morphology, cell cycle distribution, mitotic marker analyses and the number of multinucleated cells and apoptotic cells. Furthermore, MBZ treatment inhibited activation of Akt and Erk in AML leukemic cells. Finally, MBZ repressed the progression of leukemic cells in vivo and prolonged survival in AML xenograft mouse model. Taken together, our results suggest that MBZ could be a potential new therapeutic agent for the treatment of AML patients.

Targeting Androgen Receptor in Treating HER2 Positive Breast Cancer.

Androgen receptor (AR) is widely expressed in different subtypes of breast cancer (BC). However, it is unclear how AR functions in HER2 positive (+) BC. Knockdown of AR with shRNAs and a new generation anti-androgen drug, Enzalutamide, were used to explore the involvement of AR on the growth of HER2 + BC cells (HCC1954 and SKBR3). AR shRNA or Enzalutamide inhibited the growth of SKBR3 cells at a similar extend compared to trastuzumab, an approved HER2 targeted drug. Combining Enzalutamide with trastuzumab further decreased the growth of HCC1954 and SKBR3 cells compared with single agent alone in vitro. Biochemical analysis revealed that inhibiting AR resulted in decreased HER2 phosphorylation and activation of Erk and Akt, without affecting the HER2 and HER3 expression. The in vivo efficacy of Enzalutamide was further tested using the HCC1954 xenograft model. Enzalutamide impaired the growth of HCC1954 tumor at a level comparable to that by trastuzumab. Enzalutamide decreased Ki67 staining and increased activated caspase3 staining compared with vehicle control in HCC1954 tumors. Our results indicate AR plays an important role in promoting the growth of HER2 + BC by cross-talking with the HER2 signaling. AR drug may be used as an alternative second line therapy for treating HER2 + BC.

Direct and indirect gene diagnosis of hemophilia A pedigrees in the Chinese population.

Hemophilia A (HA) is an X­linked recessive hereditary disorder caused by defects in the coagulation factor VIII (FVIII) gene. In order to diagnose patients with presymptomatic HA and carriers, the present study conducted direct gene diagnosis for the common abnormalities in FVIII and subsequently performed indirect gene diagnosis for the other abnormalities in FVIII for Chinese HA pedigrees. Direct gene diagnosis was performed in 10 HA pedigrees using inverse shifting­polymerase chain reaction to detect intron 22 inversion (inv22), intron 22 deletion, intron 22 duplication and inv1 of FVIII. Pedigrees with no detected mutations were further analyzed using indirect genetic diagnosis (haplotype linkage analysis), where the genetic markers of FVIII included one variable number of tandem repeat, seven short tandem repeats and three restriction fragment length polymorphisms. The results of three pedigrees were taken as examples. Pedigree 1 underwent direct gene diagnosis, which demonstrated that the proband was inv22 distal pattern hemophiliac and the mother was an inv22 distal pattern carrier. The other two pedigrees were subjected to indirect gene diagnosis. In pedigree 2, the detection of DXS52, 13(CA) n, DXS9901(GT) n, intron (int)18, int19 and int22 confirmed the proband's baby brother was normal, the proband's maternal aunt was a carrier and her baby son was normal. Detection of DXS9901(GT)n, int18, int19 and int22 in pedigree 3 demonstrated that the proband's maternal grandmother was not a carrier. As the maternal grandfather was not affected by the disease, it was deduced that a mutation of FVIII occurred in the proband's mother. The combination of direct and indirect gene diagnoses provides reliable evidence for the use of genetic counseling in HA pedigrees, particularly for screening presymptomatic males and female carriers with normal offspring.

Curcumin suppresses gastric tumor cell growth via ROS-mediated DNA polymerase γ depletion disrupting cellular bioenergetics.

BACKGROUND: Curcumin, as a pro-apoptotic agent, is extensively studied to inhibit tumor cell growth of various tumor types. Previous work has demonstrated that curcumin inhibits cancer cell growth by targeting multiple signaling transduction and cellular processes. However, the role of curcumin in regulating cellular bioenergetic processes remains largely unknown. METHODS: Western blotting and qRT-PCR were performed to analyze the protein and mRNA level of indicated molecules, respectively. RTCA, CCK-8 assay, nude mice xenograft assay, and in vivo bioluminescence imaging were used to visualize the effects of curcmin on gastric cancer cell growth in vitro and in vivo. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate. RESULTS: Curcumin significantly inhibited gastric tumor cell growth, proliferation and colony formation. We further investigated the role of curcumin in regulating cellular redox homeostasis and demonstrated that curcumin initiated severe cellular apoptosis via disrupting mitochondrial homeostasis, thereby enhancing cellular oxidative stress in gastric cancer cells. Furthermore, curcumin dramatically decreased mtDNA content and DNA polymerase γ (POLG) which contributed to reduced mitochondrial oxygen consumption and aerobic glycolysis. We found that curcumin induced POLG depletion via ROS generation, and POLG knockdown also reduced oxidative phosphorylation (OXPHOS) activity and cellular glycolytic rate which was partially rescued by ROS scavenger NAC, indiating POLG plays an important role in the treatment of gastric cancer. Data in the nude mice model verified that curcumin treatment significantly attenuated tumor growth in vivo. Finally, POLG was up-regulated in human gastric cancer tissues and primary gastric cancer cell growth was notably suppressed due to POLG deficiency. CONCLUSIONS: Together, our data suggest a novel mechanism by which curcumin inhibited gastric tumor growth through excessive ROS generation, resulting in depletion of POLG and mtDNA, and the subsequent disruption of cellular bioenergetics.

Shp1 positively regulates EGFR signaling by controlling EGFR protein expression in mammary epithelial cells.

SH2-domain containing protein tyrosine phosphatase 1 (Shp1/PTPN6) is mainly expressed in hematopoietic cells and acts a negative signaling regulator. Although Shp1 is also expressed in epithelial cells, the function of shp1 in normal epithelial is still less well understood, especially in regulating the growth of epithelial cells. In this study, different shRNAs and siRNAs against Shp1 were used to knockdown Shp1 expression in MCF10A, an immortalized mammary epithelial cell line. Shp1 knockdown resulted in inhibited cell growth in part due to lower percentage of MCF10A cells entering into S phase and reduced cyclin D1 expression. Accordingly, EGF-induced tyrosyl phosphorylation of EGFR and Stat5 was significantly inhibited in cells with Shp1 knockdown compared with control whereas EGF-induced Akt and Erk phosphorylation was not affected by Shp1 knockdown. Further analysis revealed that Shp1 knockdown lead to decreased EGFR protein expression without affecting EGFR mRNA expression or increasing EGFR protein degradation. Our data indicate that Shp1 functions as a positive regulator and acts in a novel mechanism through promoting EGFR protein expression in mammary epithelial cells.

[Protective effects of Curcumin analogue L6H4 on kidney from type 2 diabetic rats].

OBJECTIVE: To investigate the protective effect of curcumin analogue L6H4 on the kidney from the type 2 diabetic rats. METHODS: Twenty-four SPF male SD rats were randomly divided into 3 groups(n=8):normal control group(NC),diabetes mellitus group(DM) and DM+L6H4-treatment group(DT). After rats were fed with high-fat diet for 4 weeks, both the DM and DT groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus models. The rats in DT group were given L6H4 by gavage at the dose of 0.2 mg/kg·d for 8 weeks. After the treatment, the 24 h urinary protein, fasting blood glucose (FBG), triglyceride (TG), serum creatinine(Scr),blood urea nitrogen (BUN) and uric acid (UA) were detected biochemically. The pathological changes of the kidneys were observed under light and transmission electron microscopes. The expressions of TGF-ß1, FN and Col IV were detected by immunohistochemistry. RESULTS: The levels of the 24 h urinary protein, FBG, TG, Scr and BUN were elevated significantly in diabetic group(P<0.01). The glomerular volume of DM group rats became irregularly enlarged, diffused mesangial matrix accumulated, with basal membrane proliferous hypertrophy and fusion phenomenon of foot process, the expressions of TGF-ß1,FN and Col-IV were elevated significantly (P<0.05). After treated with L6H4, the levels of the 24 h urinary protein, FBG, TG, Scr and BUN were decreased in DT group compared to DM group (P<0.01), the morphological changes of kidney were ameliorated. The expression levels of TGF-ß1, FN and Col-IV were downregulated (P<0.05). CONCLUSIONS: L6H4 exerts the protective effect on kidneys of type 2 diabetic rats by reducing expression of TGF-ß1, inhibiting secretion of Col-IV and FN, relieving the deposition of the extracellular matrix.