[Show abstract][Hide abstract] ABSTRACT: Leukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.
We constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.
Both fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.
These results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.
Full-text · Article · Dec 2015 · Journal of Hematology & Oncology
[Show abstract][Hide abstract] ABSTRACT: Background:
B-acute lymphoblastic leukemia (B-ALL) is derived from B cell progenitors. Recently, the development of appropriate combinations of chemotherapy and immunotherapy represents a promising approach for eliminating cancer. We previously constructed an anti-CD3 × anti-CD19 bi-specific antibody in a diabody configuration and its disulfide-stabilized format (ds-diabody). The combination of the diabody or ds-diabody and Ara-C was highly effective in enhancing the cytotoxicity of T cells against the CD19+ human leukemia cell-line, Nalm-6, both in vitro and in vivo. This study verified whether B-ALL patient-derived cells were sensitive to the diabody or ds-diabody and low-dosage Ara-C combination.
This study aimed to detect the B7 family members B7.1 (CD80) and B7.2 (CD86) that were expressed in B-ALL patient-derived cells pre-treated by Ara-C (0.25 μM) and to determine the targeted killing ability of T cell subtypes induced by the diabody or ds-diabody combination with Ara-C both in vitro and in vivo. We also determined the levels of the cytokines that were released by activated CD4+ or CD8+ T cells during therapy.
Low-dose Ara-C enhanced CD80 and CD86 expression in nearly 50 % of specimens of B-ALL patient-derived cells. A combination of diabody or ds-diabody and Ara-C enhanced T cell against B-ALL cells in vitro and in vivo. Both CD8+ and CD4+ T cells were potently activated. Expression of CD25 and CD69 was augmented equally by CD4+ or CD8+ T cells. However, CD8+ T cells made the major contribution by redirecting target cell lysis in a granzyme B and perforin-dependent mechanism. CD4+ T cells played an important immunomodulatory role by secreting IL2. Consequently, IL3, IL6, TNFα, and IFNγ were also released by CD4+ or CD8+ T cells following diabody-mediated T cell activation.
T cell therapy induced by diabody or ds-diabody combined with low dose of Ara-C was effective against cancer cell-lines and in clinical trials. In vivo, the ds-diabody was more efficient than its parent diabody due to its enhanced stability.
Preview · Article · Oct 2015 · Journal of Hematology & Oncology
[Show abstract][Hide abstract] ABSTRACT: Rituximab is widely used in clinical setting for the treatment of B malignant lymphoma and has achieved remarkable success. However, in most patients, the disease ultimately relapses and become resistant to rituximab. To overcome the limitation, there is still a need to find novel strategy for improving therapeutic efficacy.
To construct genetically engineered antibody anti-CD19(Fab)-LDM, and verify the anticancer activity targeted toward B-lymphoma.
The anticancer activity of anti-CD19(Fab)-LDM in vitro and in vivo was examined. In vitro, the binding activity and internalization of anti-CD19(Fab)-LDP were measured. Using comet assay and apoptosis, the cytotoxicity of energized fusion proteins was observed. From in vivo experiments, targeting of therapeutic effect and anticancer efficacy bythe fusion protein was verified.
Data showed that anti-CD19(Fab)-LDM does not only binding the cell surface but is also internalized into the cell. The energized fusion proteins anti-CD19(Fab)-LDM can induce DNA damage. Furthermore, significant in vivo therapeutic efficacy was observed.
The present study demonstrated that the genetically engineered antibody anti-CD19(Fab)-LDM exhibited enhanced cytotoxicity compared to LDM alone. One of the most powerful advantages of anti-CD19(Fab)-LDM, however, is that it can be internalized within the cells and carry out cytotoxic effects. Therefore, anti-CD19(Fab)-LDM may be as a useful targeted therapy for B-cell lymphoma.
No preview · Article · Jul 2015 · Journal of Drug Targeting
[Show abstract][Hide abstract] ABSTRACT: CD123 bacame a therapeutic target for acute myelocytic leukemia(AML) because of its overexpression only on AML stem cells.It is αsubunit of interleukin-3 (multi-CSF, IL3) receptor. Lidamycin(LDM) is a novle antibiotic composed of an apoprotein(LDP) and a chromophore(AE). We cloned, expressed and isolated IL3LDP fusion protein first then assembled with AE in vitro. We found that131/132 amino acids of IL3 were the key factors for IL3 fusion protein stability and I131L/F132L mutation effectively improved the IL3 fusion protein stability.The toxicity of IL3LDM to CD123+ tumor cells was 2-10 times compared to LDM alone and 10000 times compared to ADR. Meanwhile, IL3LDM impaired the colony-forming ability of CD123+ stem-like cells but not to CD123 negtive normal cord blood cells. Three drug delivery methods in vivo were adopted: prophylactic treatment and single/multiple-dosing administration. The tumor-free survival extended to 120 days and cancer cell invasion significantly decreased after IL3LDM continuous multiple treated. Moreover, IL3LDM had been shown to modulate apoptosis by arrested cell cycle in G2/M phase. Therefore, IL3LDM is expected to be a new drug for leukemia target therapy.
No preview · Article · Jul 2015 · Cancer biology & therapy
[Show abstract][Hide abstract] ABSTRACT: Overexpression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases, including rheumatoid arthritis,
inflammatory bowel disease, and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions.
To explore chemical inhibitors against TNFα activity, we applied computer-aided drug design combined with in vitro and cell-based assays and identified a lead chemical compound, (E)-4-(2-(4-chloro-3-nitrophenyl) (named as C87 thereafter), which directly binds to TNFα, potently inhibits TNFα-induced cytotoxicity
(IC50 = 8.73 μm) and effectively blocks TNFα-triggered signaling activities. Furthermore, by using a murine acute hepatitis model, we showed
that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival.
Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat
TNFα-mediated inflammatory diseases.
No preview · Article · Mar 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are promising vehicles for delivering therapeutic agents in tumor therapy. Human umbilical cord-derived mesenchymal stem cells (HUMSCs) resemble bone marrow-derived MSCs with respect to hepatic differentiation potential in injured livers in animals, while their hepatic differentiation under the hepatocarcinoma microenvironment is unclear. In this study, HUMSCs were isolated and transduced by lentiviral vectors coding the soluble human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) gene driven by alpha-fetoprotein (AFP) promoter to investigate the therapeutic effects of these HUMSC against orthotopically implanted hepatocarcinoma in mice. We showed that HUMSCs can be transduced by lentivirus efficiently. HUMSCs developed cuboidal morphology, and expressed AFP and albumin in a two-step protocol. HUMSCs were capable of migrating to hepatocarcinoma in vitro as well as in vivo. In the orthotopical hepatocarcinoma microenvironment, the AFP promoter was activated during the early hepatic differentiation of HUMSCs. After intravenous injected, MSC.AFPILZ-sTRAIL expressed sTRAIL exclusively at the tumor site, and exhibited significant antitumor activity. This effect was stronger when in combination with 5-FU. The treatment was tolerated well in mice. Collectively, our results provide a potential strategy for targeted tumor therapy relying on the use of the tumor tropism and specific differentiation of HUMSCs as vehicles.
[Show abstract][Hide abstract] ABSTRACT: Sorcin, a 22-kDa calcium-binding protein, renders cancer cells resistant to chemotherapeutic agents, thus playing an important role in multidrug resistance. As there is a clear association between drug resistance and an aggressive phenotype, we asked whether sorcin affects also the motility, invasion, and stem cell characteristics of cancer cells. We have used both RNA interference (transient and stable expression of hairpins) and a lentiviral expression vector to experimentally modulate sorcin expression in a variety of cells. We demonstrate that sorcin depletion in MDA-MB-231 breast cancer cells reduces the pool of CD44(+)/CD24(-) and ALDH1(high) cancer stem cells (CSCs) as well as mammosphere-forming capacity. We also observe that sorcin regulates epithelial-mesenchymal transition and CSCs partly through E-cadherin and vascular endothelial growth factor expression. This leads to the acquisition of an epithelial-like phenotype, attenuating epithelial-mesenchymal transition and suppression of metastases in nude mice. The sorcin-depleted phenotype can also be reproduced in lung adenocarcinoma A549 cells and lung fibrosarcoma HT1080 cells. In addition, overexpression of sorcin in MCF7 cells, which have low endogenous sorcin expression levels, increases their migration and invasion in vitro. This offers the rationale for the development of therapeutic strategies down-regulating sorcin expression for the treatment of cancer.
No preview · Article · Dec 2013 · Breast Cancer Research and Treatment
[Show abstract][Hide abstract] ABSTRACT: Resistance to chemotherapeutic treatment, which is indirectly responsible for many cancer deaths, is normally associated with an aggressive phenotype including increased cell motility and acquisition of invasive properties. Here we describe how breast cancer cells overcome doxorubicin-induced senescence and become drug resistant by overexpression of the microRNA (miR)-106b∼25 cluster. Although all three miRs in the cluster contribute to the generation of doxorubicin resistance, miR-25 is the major contributor to this phenotype. All three miRs in this cluster target EP300, a transcriptional activator of E-cadherin, resulting in cells acquiring a phenotype characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT), including an increase in both cell motility and invasion, as well as the ability to proliferate after treatment with doxorubicin. These findings provide a novel drug resistance/EMT regulatory pathway controlled by the miR-106b∼25 cluster by targeting a transcriptional activator of E-cadherin.Cell Death and Differentiation advance online publication, 22 November 2013; doi:10.1038/cdd.2013.167.
No preview · Article · Nov 2013 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: Sorcin, a 22-kDa calcium-binding protein, renders cancer cells resistant to chemotherapeutic agents, thus playing an important role in multidrug resistance (MDR). But the mechanisms mediated by sorcin still remain quite elusive. This study aim to explore whether sorcin silencing could restore chemosensitivity in MDR cancer cells and seek to identify the functional mechanisms mediated by sorcin.
To investigate the mechanisms of sorcin-silencing-induced chemosensitivity, transient expression of sorcin-siRNAs was performed in doxorubicin-induced MDR cell lines, K562/A02 and MCF-7/A02. Sensitivity to five chemotherapeutic agents was evaluated by analysis of cell survival and cell apoptosis.
In this report, we show that down-regulation of sorcin did not alter expression or function of P-gp, but actually induced cell apoptosis and chemosensitivity in K562/A02 and MCF-7/A02. We also observe that silencing of sorcin-enhanced chemotherapeutic agent effects partly through regulating apoptosis-related protein, including Bcl-2, Bax, c-jun and c-fos.
This offers the rationale for the development of therapeutic strategies down-regulating sorcin expression for the treatment of cancer, especially for the reversal of MDR.
No preview · Article · Sep 2013 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHⅡ-7 is a derivative of indirubin, which is designed and synthesized by our laboratory. Our preliminary work indicates its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effect in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells. However, the detailed mechanism of PHⅡ-7 remains to be explored. Reactive Oxygen Species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHⅡ-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHⅡ-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHⅡ-7. Our results collectively indicated that reactive oxygen species production induced by PHⅡ-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was induced by PHⅡ-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results provide a detailed biochemical basis for further clinical application of PHⅡ-7.
No preview · Article · Jul 2013 · European journal of pharmacology
[Show abstract][Hide abstract] ABSTRACT: Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. So the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format which is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, antiCD3-antiCD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19+ human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could up-regulate CD80 expressed on CD19+ Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by antiCD3×antiCD19 diabody with or without low dose of Ara-C was compared. The combination of antiCD3×antiCD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2 (IL-2). Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could up-regulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could up-regulate CD80 expressed on 2 of 5 specimens of acute B lymphoblastic leukemia (B-ALL) patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of antiCD3×antiCD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects.
No preview · Article · Jul 2013 · Human gene therapy
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are attractive candidate for cell-based therapy. We have designed a promising double-target therapeutic system for non-Hodgkin's lymphoma therapy. The system is based on MSC homing capacity and scFvCD20 antigen-restriction to non-Hodgkin's lymphoma (NHL). In this system, a novel secreted fusion protein scFvCD20-sTRAIL, which contains a CD20-specific single chain Fv antibody fragment (scFv) and a soluble tumor necrosis factor related apoptosis-inducing ligand (sTRAIL, aa residues 114-281) with an isoleucine zipper was added to the N-terminal (IST-sTRAIL), was expressed in human umbilical cord derived mesenchymal stem cells (HUMSCs) . When compared with IST-sTRAIL protein, the scFvCD20-sTRAIL fusion protein have demonstrated a potent inhibition of cell proliferation in CD20-positive BJAB cells, moderate inhibition in Raji cells, weak inhibition in CD20-negtive Jurkat cells and no effect on normal human peripheral blood mononuclear cells (PBMCs). The scFvCD20-sTRAIL fusion protein also caused significant increase of cellular apoptosis through both extrinsic and intrinsic apoptosis signaling pathways. Using a NOD/SCID mouse subcutaneous BJAB lymphoma xenograft model, the tropism of the firefly luciferase (fLuc) labeled MSC was monitored by bioluminescent imaging (BLI) for fLuc activity. Our study indicated that HUMSCs selectively migrated to tumor site after 24 hours of intravenous injection and mice injected with the MSC.scFvCD20-sTRAIL significantly inhibited the tumor growth when compared with those treated with MSC.IST-sTRAIL. The treatment was tolerated well in mice, as no obvious toxicities were observed. Our study has suggested that scFvCD20-sTRAIL secreting HUMSCs is a novel and efficient therapeutic approach for the treatment of non-Hodgkin's Lymphoma.
No preview · Article · Nov 2012 · Molecular Pharmaceutics
[Show abstract][Hide abstract] ABSTRACT: Microsomal cytochrome P450 (P450) enzymes, which are important in the metabolism of carcinogens, are expressed in both epithelial and stromal cells in the mammary gland. The aim of this study was to investigate the roles of mammary epithelial P450 enzymes in the bioactivation and disposition of 7,12-dimethylbenz(a)anthracene (DMBA), a breast carcinogen, in the mammary gland. A new mouse model (named MEpi-Cpr-null) was produced, wherein P450 activities in the mammary epithelial cells are suppressed through tissue-specific deletion of the gene for P450 reductase (Cpr), an enzyme required for the activities of all microsomal P450 enzymes. Comparisons between wild-type and MEpi-Cpr-null mice showed that the tissue-specific deletion of Cpr in the mammary epithelial cells was accompanied by significant increases in the levels of DMBA and DMBA-DNA adduct in the mammary gland following a single intraperitoneal injection of DMBA at 50mg/kg. Immunohistochemical and immunoblot analysis further revealed greater induction of CYP1B1 expression by the DMBA treatment in the mammary stroma of the MEpi-Cpr-null mice than in that of the WT mice. These findings not only demonstrate that the epithelial P450 enzymes play important roles in the clearance of DMBA, but also suggest that P450 enzymes in both mammary epithelial and stromal cells contribute to carcinogen-mediated DNA damage.
No preview · Article · May 2012 · Toxicology Letters
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We examined ABCB5 gene expression using real-time polymerase chain reaction (PCR) in leukemia cells from 29 patients with acute lymphoblastic leukemia (ALL), 24 patients with chronic lymphocytic leukemia (CLL), 42 with acute myeloid leukemia (AML), 22 with chronic myeloid leukemia (CML), 17 with lymphoma and 10 with multiple myeloma (MM). It was confirmed that expression of the ABCB5 gene is highly increased in B-precursor ALL and French-American-British (FAB) M1 and M2 types of AML and lymphoma. The ABCB5 gene is expressed more highly in patients with relapsed or refractory disease than in patients with drug sensitive acute leukemia. Furthermore, there was an evident positive correlation between ABCB5 mRNA expression and MDR1 mRNA expression, but no correlation with MRP mRNA expression or BCRP mRNA expression. Quantification of the ABCB5 gene by real-time PCR offers particular promise as a prognostic marker and a marker for drug resistance in acute leukemia. Our findings raise the possibility that ABCB5 may be responsible for both the progression and chemotherapeutic refractoriness of advanced acute leukemia, and that ABCB5-targeted approaches might therefore represent novel and translationally relevant therapeutic strategies for drug resistance in leukemia.
No preview · Article · Nov 2011 · Leukemia & lymphoma
[Show abstract][Hide abstract] ABSTRACT: Multidrug resistance (MDR) is a major impediment to the effective chemotherapy of many human malignancies, and novel MDR reversal agents are desirable for combination therapy to reduce MDR, enhance anti-tumor activity and reduce side effects. Overexpression of P-glycoprotein (P-gp) is the most prevalent cause of MDR in cancer tissues, and resistance to apoptosis is a common characteristic for the multidrug resistant cancer cells. Our group has synthesized a novel potent anti-tumor indirubin derivative, PHII-7. In this study, MCF-7/ADR cells, an adriamycin (ADR)-selected human breast tumor cell line with the MDR phenotype, were used to investigate the anticancer properties of this novel indirubin derivative. Cytotoxicity and apoptosis assays showed that PHII-7 significantly inhibited cell growth, induced apoptosis, potentiated ADR cytotoxicity and restored chemotherapy sensitivity in the MDR cancer cells. Further studies indicated that by down-regulation of P-gp expression, PHII-7 partially inhibited P-gp efflux pump function and increased intracellular accumulation of Rhodamine 123, a P-gp substrate. These results provide a biochemical basis for possible clinical application of PHII-7 alone or in combination with conventional antineoplastic agents in the treatment MDR tumors.
No preview · Article · Aug 2011 · European journal of pharmacology
[Show abstract][Hide abstract] ABSTRACT: Integrin αvβ3 plays a critical role in the survival and metastasis process of cancer cells. It is therefore desirable to develop new types of small molecule inhibitors of integrin αvβ3. IH1062 (3, 5-dichloro-phenylbiguanide) is a novel small molecule inhibitor of integrin αvβ3 that we have recently discovered. In this study, we investigated the induction effects of anoikis in human melanoma cell line M21 by IH1062, by detecting caspase activity, measuring the expression levels of apoptosis-related proteins, and performing the AnnexinV/PI apoptosis assay. Furthermore, we established a melanoma pulmonary metastasis mouse model in order to evaluate the suppression of metastasis by IH1062 in vivo. Our results demonstrate that IH1062 triggered human melanoma M21 cells to undergo anoikis by interrupting the attachment of M21 cells to extracellular matrix, reducing the phosphorylation of focal adhesion kinase, decreasing survivin and the ratio of Bcl-2/Bax proteins, and activating caspase cascades in vitro. Additionally, IH1062 showed markedly anti-metastatic effects in the pulmonary metastasis model in vivo, which makes it a promising lead to develop new drugs for anti-metastasis therapies.
No preview · Article · Aug 2011 · Investigational New Drugs