Yingdai Gao

Synthesis of a Dual Functional Anti-MDR Tumor Agent PH II-7 with Elucidations of Anti-Tumor Effects and Mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux.

Improvement of tumor targeting and antitumor activity by a disulphide bond stabilized diabody expressed in Escherichia coli

We have generated an anti-Pgp/anti-CD3 diabody which can effectively inhibit the growth of multidrug-resistant human tumors. However, the two chains of the diabody are associated non-covalently and are therefore capable of dissociation. Cysteine residues were introduced into the V-domains to promote disulphide cross-linking of the dimer as secreted by Escherichia coli. Compared with the parent diabody, the ds-Diabody obtained was more stable in human serum at 37°C, without loss of affinity or cytotoxicity activity in vitro. Furthermore, the ds-Diabody showed improved tumor localization and a twofold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts. Our data demonstrate that ds-Diabody may be more useful in therapeutic applications than the parent diabody.

Extracellular domain of 4-1BBL enhanced the antitumoral efficacy of peripheral blood lymphocytes mediated by anti-CD3 × anti-Pgp bispecific diabody against human multidrug-resistant leukemia

Our previous data have shown a significantly higher tumor response to anti-CD3/anti-Pgp bispecific diabody-mediated immunotherapy for P-glycoprotein (Pgp)-overexpressing K562/A02 cells, but a rapid tumor relapse occurred at 1 week after therapy. In an attempt to overcome tumor recurrence, we supplemented the previous therapy with extracellular domain of human 4-1BBL (ex4-1BBL) to regulate the activation of peripheral blood lymphocyte (PBL). As a result, this combination showed enhanced cytotoxicity in vitro and eradicated the multidrug-resistant xenografts of K562/A02 in nude mice. Furthermore, no tumor recurrence was observed within 100 days after the first treatment. Therefore, when used as an adjuvant, ex4-1BBL may improve the outcome of PBL-based immunotherapy.

Upregulation of miR-99a is associated with poor prognosis of acute myeloid leukemia and promotes myeloid leukemia cell expansion

Leukemia stem cells (LSCs) can resist available treatments that results in disease progression and/or relapse. To dissect the microRNA (miRNA) expression signature of relapse in acute myeloid leukemia (AML), miRNA array analysis was performed using enriched LSCs from paired bone marrow samples of an AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a was upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients, and higher expression levels of miR-99a were significantly correlated with worse overall survival and event-free survival in these AML patients. Ectopic expression of miR-99a led to increased colony forming ability and expansion in myeloid leukemia cells after exposure to chemotherapeutic drugs in vitro and in vivo, partially due to overcoming of chemotherapeutic agent-mediated cell cycle arrest. Gene profiling and bioinformatic analyses indicated that ectopic expression of miR-99a significantly upregulated genes that are critical for LSC maintenance, cell cycle, and downstream targets of E2F and MYC. This study suggests that miR-99a has a novel role and potential use as a biomarker in myeloid leukemia progression.

Antineoplastic effects and mechanisms of micheliolide in acute myelogenous leukemia stem cells

Leukemic stem cells (LSCs) greatly contribute to the initiation, relapse, and multidrug resistance of leukemia. Current therapies targeting the cell cycle and rapidly growing leukemic cells, including conventional chemotherapy, have little effect due to the self-renewal and differentiated malignant cells replenishment ability of LSCs despite their scarce supply in the bone marrow. Micheliolide (MCL) is a natural guaianolide sesquiterpene lactone (GSL) which was discovered in michelia compressa and michelia champaca plants, and has been shown to exert selective cytotoxic effects on CD34+CD38− LSCs. In this study, we demonstrate that DMAMCL significantly prolongs the lifespan of a mouse model of human acute myelogenous leukemia (AML). Mechanistic investigations further revealed that MCL exerted its cytotoxic effects via inhibition of NF-κB expression and activity, and by generating intracellular reactive oxygen species (ROS). These results provide valuable insight into the mechanisms underlying MCL-induced cytotoxicity of LSCs, and support further preclinical investigations of MCL-related therapies for the treatment of AML.

Antineoplastic effects of CPPTL via the ROS/JNK pathway in acute myeloid leukemia

Drug resistance and human leukocyte antigen (HLA) matching limit conventional treatment of acute myeloid leukemia (AML). Although several small molecule drugs are clinically used, single drug administration is not sufficient to cure AML, which has a high molecular diversity. Metabolic homeostasis plays a key role in determining cellular fate. Appropriate levels of reactive oxygen species (ROS) maintain the redox system balance, and excessive amounts of ROS cause oxidative damage, thus providing a strategy to eliminate cancer cells. CPPTL is a novel analogue of parthenolide that exhibited significant cytotoxicity to AML cells in vitro and induced apoptosis in a dose-dependent manner. Additionally, CPPTLs prodrug DMA-CPPTL decreased the burden of AML engraftment and prolonged survival in a mouse model administered human primary AML cells in vivo. CPPTL induced apoptosis of AML cells by stimulating ROS production, and accumulation of ROS then activated the JNK pathway, thereby promoting mitochondrial damage. These results demonstrated that CPPTL effectively eradicated AML cells in vitro and in vivo and suggested that CPPTL may be a novel candidate for auxiliary AML therapy.