Chuan-Yuan Li

Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy

In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2 (PGE2), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death–induced tumor repopulation pathway in which caspase 3 has a major role.

Apoptotic Cells Activate the “Phoenix Rising” Pathway to Promote Wound Healing and Tissue Regeneration

Caspases in dying cells trigger the release of growth signals that promote tissue repair. Rising from the Dead Tissue injury causes cell death. Apoptotic cell death enables the elimination of damaged cells, clearing the way for tissue regeneration. Enzymes called caspases are activated in apoptotic cells, and Li et al. show that in addition to helping to execute the death process, caspases 3 and 7 stimulate the release of proliferation signals from dying cells. The authors found that skin wound healing and liver regeneration were compromised in mice deficient in caspase 3 or 7. Furthermore, stem or progenitor cells injected with or without irradiated cells or injected into irradiated or undamaged tissue proliferated more in the presence of irradiated cells or tissue. The authors dub the molecular events associated with death-induced proliferation the “phoenix rising” pathway, which involves the caspase-mediated activation of phospholipase A2 and the subsequent production and release of the lipid signal prostaglandin E2, a stimulator of cell proliferation. The ability to regenerate damaged tissues is a common characteristic of multicellular organisms. We report a role for apoptotic cell death in promoting wound healing and tissue regeneration in mice. Apoptotic cells released growth signals that stimulated the proliferation of progenitor or stem cells. Key players in this process were caspases 3 and 7, proteases activated during the execution phase of apoptosis that contribute to cell death. Mice lacking either of these caspases were deficient in skin wound healing and in liver regeneration. Prostaglandin E2, a promoter of stem or progenitor cell proliferation and tissue regeneration, acted downstream of the caspases. We propose to call the pathway by which executioner caspases in apoptotic cells promote wound healing and tissue regeneration in multicellular organisms the “phoenix rising” pathway.

Enhancement of Hypoxia-Induced Tumor Cell Death In vitro and Radiation Therapy In vivo by Use of Small Interfering RNA Targeted to Hypoxia-Inducible Factor-1α

Hypoxia-inducible factor-1α (HIF-1α) is an important transcriptional factor that is activated when mammalian cells experience hypoxia, a tumor microenvironmental condition that plays pivotal roles in tumor progression and treatment. In this study, we examined the idea of down-regulating HIF-1α in tumor cells for therapeutic gain. We show that the expression levels of HIF-1α can be significantly attenuated by use of the recently established small interfering RNA technology in combination with adenovirus-mediated gene transfer. Down-regulation of the HIF-1α protein enhanced hypoxia-mediated tumor cell apoptosis in vitro. Subcutaneous tumor growth was also prevented from cells with attenuated HIF-1α expression. In addition, intratumoral injection of adenovirus encoding the HIF-1α-targeted small interfering RNA had a small but significant effect on tumor growth when combined with ionizing radiation. Therefore, our results provide proof of HIF-1α as an effective target for anticancer therapy. They also suggest that an adenovirus-based small interfering RNA gene transfer approach may be a potentially effective adjuvant strategy for cancer treatment.

Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells

Transplantation of exogenous dopaminergic neuron (DA neurons) is a promising approach for treating Parkinsons disease (PD). However, a major stumbling block has been the lack of a reliable source of donor DA neurons. Here we show that a combination of five transcriptional factors Mash1, Ngn2, Sox2, Nurr1, and Pitx3 can directly and effectively reprogram human fibroblasts into DA neuron-like cells. The reprogrammed cells stained positive for various markers for DA neurons. They also showed characteristic DA uptake and production properties. Moreover, they exhibited DA neuron-specific electrophysiological profiles. Finally, they provided symptomatic relief in a rat PD model. Therefore, our directly reprogrammed DA neuron-like cells are a promising source of cell-replacement therapy for PD.

GRP94 (gp96) and GRP94 N-Terminal Geldanamycin Binding Domain Elicit Tissue Nonrestricted Tumor Suppression

In chemical carcinogenesis models, GRP94 (gp96) elicits tumor-specific protective immunity. The tumor specificity of this response is thought to reflect immune responses to GRP94-bound peptide antigens, the cohort of which uniquely identifies the GRP94 tissue of origin. In this study, we examined the apparent tissue restriction of GRP94-elicited protective immunity in a 4T1 mammary carcinoma model. We report that the vaccination of BALB/c mice with irradiated fibroblasts expressing a secretory form of GRP94 markedly suppressed 4T1 tumor growth and metastasis. In addition, vaccination with irradiated cells secreting the GRP94 NH2-terminal geldanamycin-binding domain (NTD), a region lacking canonical peptide-binding motifs, yielded a similar suppression of tumor growth and metastatic progression. Conditioned media from cultures of GRP94 or GRP94 NTD-secreting fibroblasts elicited the up-regulation of major histocompatibility complex class II and CD86 in dendritic cell cultures, consistent with a natural adjuvant function for GRP94 and the GRP94 NTD. Based on these findings, we propose that GRP94-elicited tumor suppression can occur independent of the GRP94 tissue of origin and suggest a primary role for GRP4 natural adjuvant function in antitumor immune responses.

GW112, A Novel Antiapoptotic Protein That Promotes Tumor Growth

GW112 is a novel gene that has little homology to other known genes. It is overexpressed in a number of human tumor types, especially in those of the digestive system. We show here that GW112 is associated with GRIM-19, a protein known to be involved in regulating cellular apoptosis. Functionally, GW112 could significantly attenuate the ability of GRIM19 to mediate retinoic acid-IFN-β-mediated cellular apoptosis and apoptosis-related gene expression. In addition, GW112 demonstrated strong antiapoptotic effects in tumor cells treated with other stress exposures such as hydrogen peroxide. Finally, forced overexpression of GW112 in murine prostate tumor cells led to more rapid tumor formation in a syngeneic host. Taken together, our data suggest that GW112 is an important regulator of cell death that plays important roles in tumor cell survival and tumor growth.

Tumor Necrosis Factor-{alpha} Is a Potent Endogenous Mutagen that Promotes Cellular Transformation

Tumor necrosis factor-alpha (TNF-alpha) is an important inflammation cytokine without known direct effect on DNA. In this study, we found that TNF-alpha can cause DNA damages through reactive oxygen species. The mutagenic effect of TNF-alpha is comparable with that of ionizing radiation. TNF-alpha treatment in cultured cells resulted in increased gene mutations, gene amplification, micronuclei formation, and chromosomal instability. Antioxidants significantly reduced TNF-alpha-induced genetic damage. TNF-alpha also induced oxidative stress and nucleotide damages in mouse tissues in vivo. Moreover, TNF-alpha treatment alone led to increased malignant transformation of mouse embryo fibroblasts, which could be partially suppressed by antioxidants. As TNF-alpha is involved in chronic inflammatory diseases, such as chronic hepatitis, ulcerative colitis, and chronic skin ulcers, and these diseases predispose the patients to cancer development, our results suggest a novel pathway through which TNF-alpha promotes cancer development through induction of gene mutations, in addition to the previously reported mechanisms, in which nuclear factor-kappaB activation was implicated.

Noninvasive visualization of tumors in rodent dorsal skin window chambers

A novel model for evaluating anti-cancer therapies.

Systemic overexpression of angiopoietin-2 promotes tumor microvessel regression and inhibits angiogenesis and tumor growth

Angiopoietin-2 (Ang-2) is a conditional antagonist and agonist for the endothelium-specific Tie-2 receptor. Although endogenous Ang-2 cooperates with vascular endothelial growth factor (VEGF) to protect tumor endothelial cells, the effect on tumor vasculature of high levels of exogenous Ang-2 with different levels of VEGF has not been studied in detail. Here, we report that systemic overexpression of Ang-2 leads to unexpected massive tumor vessel regression within 24 h, even without concomitant inhibition of VEGF. By impairing pericyte coverage of the tumor vasculature, Ang-2 destabilizes the tumor vascular bed while improving perfusion in surviving tumor vessels. Ang-2 overexpression transiently exacerbates tumor hypoxia without affecting ATP levels. Although sustained systemic Ang-2 overexpression does not affect tumor hypoxia and proliferation, it significantly inhibits tumor angiogenesis, promotes tumor apoptosis, and suppresses tumor growth. The similar antitumoral, antiangiogenic efficacy of systemic overexpression of Ang-2, soluble VEGF receptor-1, and the combination of both suggests that concomitant VEGF inhibition is not required for Ang-2-induced tumor vessel regression and growth delay. This study shows the important roles of Ang-2-induced pericyte dropout during tumor vessel regression. It also reveals that elevated Ang-2 levels have profound pleiotropic effects on tumor vessel structure, perfusion, oxygenation, and apoptosis.

Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery.

Interstitial transport is a crucial step in plasmid DNA-based gene therapy. However, interstitial diffusion of large nucleic acids is prohibitively slow. Therefore, we proposed to facilitate interstitial transport of DNA via pulsed electric fields. To test the feasibility of this approach to gene delivery, we developed an ex vivo technique to quantify the magnitude of DNA movement due to pulsed electric fields in two tumor tissues: B16.F10 (a mouse melanoma) and 4T1 (a mouse mammary carcinoma). When the pulse duration and strength were 50 ms and 233 V/cm, respectively, we found that the average plasmid DNA movements per 10 pulses were 1.47 μm and 0.35 μm in B16.F10 and 4T1 tumors, respectively. The average plasmid DNA movements could be approximately tripled, ie to reach 3.69 μm and 1.01 μm, respectively, when the pulse strength was increased to 465 V/cm. The plasmid DNA mobility was correlated with the tumor collagen content, which was approximately eight times greater in 4T1 than in B16.F10 tumors. These data suggest that electric field can be a powerful driving force for improving interstitial transport of DNA during gene delivery.