Edna Mora

Sustained Small Interfering RNA Delivery by Mesoporous Silicon Particles

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA). Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma. Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the ανβ3 integrin–positive tumor endothelial cells in the A2780 tumor-bearing mice. This approach resulted in significant inhibition of tumor growth compared with controls. Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for siRNA with the potential for broad applications in human disease. Clin Cancer Res; 16(15); 3910–22. ©2010 AACR.

Orthomolecular Oncology Review: Ascorbic Acid and Cancer 25 Years Later

The effect of ascorbic acid on cancer has been a subject of great controversy. This is a follow-up review of the 1979 article by Cameron, Pauling, and Leibovitz published in Cancer Research. In this updated version, the authors address general aspects of ascorbic acid and cancer that have been presented before, while reviewing, analyzing, and updating new existing literature on the subject. In addition, they present and discuss their own mechanistic hypothesis on the effect of ascorbic acid on the cancer cell. The objective of this review is to provide an updated scientific basis for the use of ascorbic acid, especially intravenously as adjuvant treatment in pharmacological nutritional oncology.

Biological roles of the Delta family notch ligand Dll4 in tumor and endothelial cells in ovarian cancer

Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment.

Chitosan hydrogel for localized gene silencing

Objective: To achieve effective delivery of siRNA into target cells in vivo, we have developed a novel approach of siRNA delivery by using local drug delivery systems. Results: The chitosan hydrogel (CH-HG) displayed a liquid-solid phase transition in a temperature-dependent manner and formed an endothermic hydrogel in tumor tissue after intra-tumoral injection. Additionally, we tested the extent of in vivo delivery following a single intra-tumoral injection of Alexa555 siRNA/CH-HG into A375SM-bearing mice. The Alexa555 siRNA demonstrated higher localization into tumor cells compared to control. The Alexa555 siRNA delivery extends to tumor cells outside of CH-HG and some tumor cells also infiltrated into CH-HG. For therapeutic proof-of-concept studies, CH-HG including TG2-targeted siRNA significantly inhibited tumor growth in melanoma (A375SM) and breast (MDA-MB231) tumor models compared to control (A375SM: 72% reduction and MDA-MB231: 92% reduction, p<0.001). Experimental Design: we prepared a CH-HG system loaded with siRNA to enhance localized therapeutic efficacy without risk for systemic side effects. Delivery of siRNA into CH-HG was confirmed by fluorescence microscopy. Antitumor efficacy was examined in mouse models of melanoma (A375SM) and breast (MDA-MD231) cancer. Conclusions: This study developed a novel local delivery method for siRNA therapy using the CH-HG system. This approach could have broad applications for multiple localized diseases. See commentary: Hydrogel-siRNA for cancer therapy

Erythropoietin Stimulates Tumor Growth via EphB4

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpos effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Aggressive therapy for locoregional recurrence after mastectomy in stage II and III breast cancer patients

AbstractBackground: To determine if aggressive treatment of locoregional recurrence affects survival, we retrospectively analyzed the clinical outcome of 69 breast cancer patients who developed locoregional disease as their first episode of recurrence following mastectomy and adjuvant chemotherapy. Methods: Patients were identified from among 1,707 stage II and III breast cancer patients who enrolled in five different doxorubicin-based adjuvant chemotherapy protocols at The University of Texas M. D. Anderson Cancer Center from 1975 to 1986. Sixty-nine evaluable patients who had a locoregional recurrence as the first site of relapse after mastectomy formed the study group. Multifactorial analysis of clinical and histopathological characteristics of both the primary tumor and the subsequent recurrence was performed using a logistic regression method. Survival analysis was performed using an actuarial life-table method calculated from the date of registration into the adjuvant therapy protocols. Results: Median follow-up was 6.6 years. Two factors significantly affected survival: recurrence of disease during or after adjuvant treatment of the primary and whether the patient was rendered disease free after recurrence. Conclusions: Stage II and III breast cancer patients who have locoregional recurrence after adjuvant chemotherapy and can be rendered disease free may have a better survival rate. Aggressive treatment of locoregional recurrence including complete surgical excision should be considered in this subgroup of patients.

Synthesis, stability, cellular uptake, and blood circulation time of carboxymethyl-inulin coated magnetic nanoparticles

Iron oxide nanoparticles were coated with the biocompatible, biodegradable, non-immunogenic polysaccharide inulin by introduction of carboxyl groups into the inulin structure and conjugation with amine groups on the surface of iron oxide nanoparticles grafted with 3-aminopropyltriethoxysilane. The resulting nanoparticles were characterized by FT-IR spectroscopy, transmission electron microscopy, dynamic light scattering, zeta potential, SQUID magnetometry, and with respect to their energy dissipation rate in applied alternating magnetic fields. The nanoparticles had a hydrodynamic diameter in the range of 70 ± 10 nm and were superparamagnetic, with energy dissipation rates in the range of 58-175 W/g for an applied field frequency of 233 kHz and an applied field amplitude in the range of 20-48 kA/m. The nanoparticles were stable in a range of pH, at temperatures between 23°C and 53°C, and in short term storage in water, PBS, and culture media. The particles were non-cytotoxic to the immortalized human cancer cell lines Hey A8 FDR, A2780, MDA 468, MCF-7 and Caco-2. The nanoparticles were readily taken up by Caco-2 cells in a time and concentration dependent fashion, and were found to have a pharmacokinetic time constant of 47 ± 3 min. The small size, non-cytotoxicity, and efficient energy dissipation of the particles could make them useful for biomedical applications such as magnetic fluid hyperthermia.

Differential motility stimulation but not growth stimulation or adhesion of metastatic human colorectal carcinoma cells by target organ-derived liver sinusoidal endothelial cells.

Liver is the most common distant metastatic site for colorectal cancers and when blood-borne colorectal cancer cells reach the liver, they first encounter hepatic capillary and sinusoidal endothelial cells. Thus we studied differences between highly (HT-29LMM) and poorly (HT-29P) liver-metastatic sublines of human colorectal cancer cells by examining the interactions between tumor cells and liver microvessel endothelial cells. Using hepatic sinusoidal endothelial (HSE) and lung microvessel endothelial (MLE) cell-conditioned medium we measured the growth and motility stimulating activities released from these endothelial cells and adhesion of these cancer cells to the endothelial cells. Differences in the ability of HSE-conditioned medium (HSE-CM) or MLE-conditioned medium (MLE-CM) to stimulate HT-29 cell growth were not observed. There was a small but significant increase in the rate of adhesion of highly metastatic HT-29LMM cells to HSE cell monolayers than poorly metastatic HT-29P cells, but there was no difference in adhesion to MLE cell monolayers. HSE-CM stimulated the motility of highly metastatic colorectal cancer cells to a greater extent than the poorly metastatic cells. Motility-stimulating activity for the colorectal cancer cell lines was not detected in MLE-CM. The HSE-CM motility-stimulating activity for human HT-29 cells was not removed using antibodies against hepatocyte growth factor (HGF/SF), complement component C3 or laminin, indicating that it is not related to these known liver-derived motility factors. The results suggest that the ability of highly metastatic HT-29LMM colorectal cancer cells to colonize the liver is related to their ability to respond to liver sinusoidal endothelial cell-derived motility factors and to a lesser degree to adhere to liver sinusoidal endothelial cells.