Yan Fan

CD44 antibody-targeted liposomal nanoparticles for molecular imaging and therapy of hepatocellular carcinoma

Most hepatocellular carcinoma (HCC) therapies fail to target cancer stem cells (CSCs) and monitor cancer progression or regression. The purpose of this study was to evaluate the possibility of cancer imaging and simultaneously monitoring targeted therapy in a single animal by anti-CD44 antibody-mediated liposomal nanoparticle. In this study, an in situ liver tumor model was applied for therapy by injecting 1.0 × 10(6) HepG2 cells carrying a reporter system encoding a double fusion (DF) reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into the liver of NOD/SCID mice. A strategy was developed which specifically targeted HCC via anti-CD44 antibody-mediated liposomal nanoparticle delivery, loaded of either doxorubicin (Dox) or a triple fusion (TF) gene containing the herpes simplex virus truncated thymidine kinase (HSV-ttk) and renilla luciferase (Rluc) and red fluorescent protein (RFP). The NOD/SCID mice were subsequently treated with ganciclovir (GCV) and the growth status of tumor was monitored by optical bioluminescence imaging (BLI) of Fluc and specific targeting of the liposomal nanoparticle was tracked by Rluc imaging. CD44 antibody-mediated liposomal nanoparticle, loaded of TF plasmids, were shown to be useful for monitoring and evaluating targeting efficacy and gene therapy by non-invasive molecular imaging. Here, we demonstrate the time intensive preclinical steps involved in molecular target identification, validation, and characterization by dual molecular imaging. This targeted and traceable therapeutic strategy has potential advantages to overcome the problems of conventional tumor therapy and may open a new application for the treatment of HCC by targeting CSCs.

Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation.

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However, teratoma formation can be a major obstacle for hES‐mediated cell therapy. Therefore, tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study, hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology, we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover, FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA‐4, and the subcultured SSEA‐4+ cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA‐4+ cells derived from teratoma exhibited multipotency, retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. J. Cell. Biochem. 112: 840–848, 2011.

Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment

The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes’ expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.

Human embryonic stem cell-derived endothelial cells as cellular delivery vehicles for treatment of metastatic breast cancer.

Endothelial progenitor cells (EPCs) have shown tropism towards primary tumors or metastases and are thus potential vehicles for targeting tumor therapy. However, the source of adult EPCs is limited, which highlights the need for a consistent and renewable source of endothelial cells for clinical applications. Here, we investigated the potential of human embryonic stem cell-derived endothelial cells (hESC-ECs) as cellular delivery vehicles for therapy of metastatic breast cancer. In order to provide an initial assessment of the therapeutic potency of hESC-ECs, we treated human breast cancer MDA-MB-231 cells with hESC-EC conditioned medium (EC-CM) in vitro. The results showed that hESC-ECs could suppress the Wnt/β-catenin signaling pathway and thereby inhibit the proliferation and migration of MDA-MB-231 cells. To track and evaluate the possibility of hESC-EC-employed therapy, we employed the bioluminescence imaging (BLI) technology. To study the therapeutic potential of hESC-ECs, we established lung metastasis models by intravenous injection of MDA-MB-231 cells labeled with firefly luciferase (Fluc) and green fluorescent protein (GFP) to NOD/SCID mice. In mice with lung metastases, we injected hESC-ECs armed with herpes simplex virus truncated thymidine kinase (HSV-ttk) intravenously on days 11, 16, 21, and 26 after MDA-MB-231 cell injection. The NOD/SCID mice were subsequently treated with ganciclovir (GCV), and the growth status of tumor was monitored by Fluc imaging. We found that MDA-MB-231 tumors were significantly inhibited by intravenously injected hESC-ECs. The tumor-suppressive effects of the hESC-ECs, by inhibiting Wnt/β-catenin signaling pathway and inducing tumor cell death through bystander effect in human metastatic breast cancer model, provide previously unexplored therapeutic modalities for cancer treatment.

The Phenotypic Fate of Bone Marrow-Derived Stem Cells in Acute Kidney Injury

Background: Despite increasing attention on the role of bone marrow derived stem cells in repair or rejuvenation of tissues and organs, cellular mechanisms of such cell-based therapy remain poorly understood. Methods: We reconstituted hematopoiesis in recipient C57BL/6J mice by transplanting syngeneic GFP+ bone marrow (BM) cells. Subsequently, the recipients received subcutaneous injection of granulocyte-colony stimulating factor (G-CSF) and were subjected to acute renal ischemic injury. Flow cytometry and immunostaining were performed at various time points to assess engraftment and phenotype of BM derived stem cells. Results: Administration of G-CSF increased the release of BM derived stem cells into circulation and enhanced the ensuing recruitment of BM derived stem cells into injured kidney. During the second month post injury, migrated BM derived stem cells lost hematopoietic phenotype (CD45) but maintained the expression of other markers (Sca-1, CD133 and CD44), suggesting their potential of transdifferentiation into renal stem cells. Moreover, G-CSF treatment enhanced the phenotypic conversion. Conclusion: Our work depicted a time-course dependent transition of phenotypic characteristics of BM derived stem cells, demonstrated the existence of BM derived stem cells in damaged kidney and revealed the effects of G-CSF on cell transdifferentiation.

Discovery of a highly potent, selective and novel CDK9 inhibitor as an anticancer drug candidate

A series of novel hybrid structure derivatives, containing both LEE011 and Cabozantinib pharmacophore, were designed, synthesized and evaluated. Surprisingly, a compound 4d was discovered that highly exhibited effective and selective activity of CDK9 inhibition with IC50=12nM. It effectively induced apoptosis in breast and lung cancer cell lines at nanomolar level. Molecular docking of 4d to ATP binding site of CDK9 kinase demonstrated a new hydrogen bonding between F atom of 4-(3-fluorobenzyloxy) group and ASN116 residue, compared with the positive control, LEE011. The compound 4d could block the cell cycle both in G0/G1 and G2/M phase to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with compound 4d showed significant suppression of cancer with low toxicity. Taken together, this novel compound 4d could be a promising drug candidate for clinical application.

Highly Selective, Potent, and Oral mTOR Inhibitor for Treatment of Cancer as Autophagy Inducer

On the basis of novel pyrazino[2,3-c]quinolin-2(1H)-one scaffold, we designed and identified a highly selective, potent and oral mTOR inhibitor, 9m. Compound 9m showed low nanomolar activity against mTOR (IC50 = 7 nM) and greater selectivity over the related PIKK family kinases, which demonstrated only modest activity against 3 out of the 409 protein kinases. In vitro assays, compound 9m exhibited high potency against human breast and cervical cancer cells and induced tumor cell cycle arrest and autophagy. 9m inhibited cellular phosphorylation of mTORC1 (pS6 and p4E-BP1) and mTORC2 (pAKT (S473)) substrates. In T-47D xenograft mouse model, oral administration of compound 9m led to significant tumor regression without obvious toxicity. In addition, this compound showed good pharmacokinetics. Collectively, due to its high potency and selectivity, compound 9m could be used as a mTOR drug candidate.

Discovery of N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)-N8-hydroxyoctanediamide as a Novel Inhibitor Targeting Cyclin-dependent Kinase 4/9 (CDK4/9) and Histone Deacetlyase1 (HDAC1) against Malignant Cancer

A series of novel, highly potent, selective inhibitors targeting both CDK4/9 and HDAC1 have been designed and synthesized. N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H-pyrrolo[2,3- d] pyrimidin-2-yl)amino)phenyl)- N8-hydroxyoctanediamide (6e) was discovered. The lead compound 6e with excellent CDK4/9 and HDAC1 inhibitory activity of IC50 = 8.8, 12, and 2.2 nM, respectively, can effectively induce apoptosis of cancer cell lines. The kinase profiling of compound 6e showed excellent selectivity and specificity. Compound 6e induces G2/M arrest in high concentration and G0/G1 arrest in low concentration to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with 6e showed significant antitumor efficacy. The insight into mechanisms of 6e indicated that it could induce cancer cell death via cell apoptosis based on CDK4/9 and HDAC1 repression and phosphorylation of p53. Our data demonstrated the novel compound 6e could be a promising drug candidate for cancer therapy.

Novel hybrid molecule overcomes the limited response of solid tumours to HDAC inhibitors via suppressing JAK1-STAT3-BCL2 signalling

Despite initial progress in preclinical models, most known histone deacetylase inhibitors (HDACis) used as a single agent have failed to show clinical benefits in nearly all types of solid tumours. Hence, the efficacy of HDACis in solid tumours remains uncertain. Herein, we developed a hybrid HDAC inhibitor that sensitized solid tumours to HDAC-targeted treatment. Methods: A hybrid molecule, Roxyl-zhc-84 was designed and synthesized with novel architecture. The pharmacokinetics and toxicity of Roxyl-zhc-84 were analysed. The antitumour effects of Roxyl-zhc-84 on solid tumours were investigated by assessing cell growth, apoptosis and cell cycle in vitro and in three in vivo mouse models and compared to those of corresponding control inhibitors alone or in combination. Gene set enrichment analysis was performed, and relevant JAK1-STAT3-BCL2 signalling was identified in vitro and in vivo in mechanistic studies. Results: Roxyl-zhc-84 showed excellent pharmacokinetics and low toxicity. The novel hybrid inhibitor Roxyl-zhc-84 induced cell apoptosis and G1-phase arrest in breast cancer and ovarian cancer cell lines. In three mouse models, oral administration of Roxyl-zhc-84 led to significant tumour regression without obvious toxicity. Moreover, Roxyl-zhc-84 dramatically improved the limited response of traditional HDAC inhibitors in solid tumours via overcoming JAK1-STAT3-BCL2-mediated drug resistance. Roxyl-zhc-84 treatment exhibited vastly superior efficacy than the combination of HDAC and JAK1 inhibitors both in vitro and in vivo. Conclusion: Concurrent inhibition of HDAC and CDK using Roxyl-zhc-84 with additional JAK1 targeting resolved the limited response of traditional HDAC inhibitors in solid tumours via overcoming JAK1-STAT3-BCL2-mediated drug resistance, providing a rational multi-target treatment to sensitize solid tumours to HDACi therapy.

Selective and novel cyclin-dependent kinases 4 inhibitor: synthesis and biological evaluation

A series of novel LEE011 derivatives containing pyridine N-oxide were designed, synthesized and evaluated. Systematic study of the structure-activity relationship (SAR) improves the selectivity for CDK4 and led to the identification of compound 9a. The compound showed comparable CDK4 kinase activity with ribociclib and greater selectivity over the closely related CDK6 kinase. The selective CDK4 inhibitor 9a has been demonstrated the antitumor activity via G1 phase cell cycle arrest, as well as dual CDK4/CDK6 inhibitor ribociclib and significantly down-regulated the activity of CDK4-cyclinD-Rb pathway of tumor cells. Taken together, this compound may act as promising lead compound for further development of new CDK4 inhibitors.