siRNA Nanoparticle Targeting PD-L1 Activates Tumor Immunity and Abrogates Pancreatic Cancer Growth in Humanized Preclinical Model.

Pancreatic cancer is characterized by late detection, frequent drug resistance, and a highly metastatic nature, leading to poor prognosis. Antibody-based immunotherapy showed limited success for pancreatic cancer, partly owing to the low delivery rate of the drug into the tumor. Herein, we describe a poly(lactic-co-glycolic acid;PLGA)-based siRNA nanoparticle targeting PD-L1 (siPD-L1@PLGA). The siPD-L1@PLGA exhibited efficient knockdown of PD-L1 in cancer cells, without affecting the cell viability up to 6 mg/mL. Further, 99.2% of PDAC cells uptake the nanoparticle and successfully blocked the IFN-gamma-mediated PD-L1 induction. Consistently, the siPD-L1@PLGA sensitized cancer cells to antigen-specific immune cells, as exemplified by Ovalbumin-targeting T cells. To evaluate its efficacy in vivo, we adopted a pancreatic PDX model in humanized mice, generated by grafting CD34+ hematopoeitic stem cells onto NSG mice. The siPD-L1@PLGA significantly suppressed pancreatic tumor growth in this model with upregulated IFN-gamma positive CD8 T cells, leading to more apoptotic tumor cells. Multiplex immunofluorescence analysis exhibited comparable immune cell compositions in control and siPD-L1@PLGA-treated tumors. However, we found higher Granzyme B expression in the siPD-L1@PLGA-treated tumors, suggesting higher activity of NK or cytotoxic T cells. Based on these results, we propose the application of siPD-L1@PLGA as an immunotherapeutic agent for pancreatic cancer.

Systemic delivery of CRISPR/Cas9 to hepatic tumors for cancer treatment using altered tropism of lentiviral vector.

Therapeutic application of CRISPR/Cas9 nucleases remains a challenge due to the lack of efficient in vivo delivery carriers. Here, we examine the ability of lentiviral vectors pseudotyped with hepatitis C virus (HCV)/E1E2 envelope glycoproteins to systemically deliver CRISPR/Cas9 to hepatic tumors in vivo. We demonstrated that systemic administration of E1E2-pseudotyped lentiviral vectors can selectively deliver Cas9 and sgRNA specific for kinesin spindle protein (KSP) to Huh7 tumors in the orthotopic Huh7 mice due to the specific interactions between E1E2 and their cellular receptors. This specific delivery leads to effective KSP gene disruption, potently inhibiting tumor growth. Furthermore, we demonstrated that E1E2-pseudotyping is more suitable for systemic delivery of CRISPR/Cas9 in cancer therapy than vesicular stomatitis virus-pseudotyping, the most widely used pseudotyping, because of stability in human serum, little transduction to DCs, low innate immune response, and cell-specific targeting ability. This study suggests that E1E2-pseudotyped lentivirus carrying CRISPR/Cas9 can substantially benefit the treatment of Huh7 tumors.

Preclinical assessment of the VEGFR inhibitor axitinib as a therapeutic agent for epithelial ovarian cancer.

Axitinib, small molecule tyrosine kinase inhibitor, demonstrates anti-cancer activity for various solid tumors. We investigated anti-cancer effect of axitinib in epithelial ovarian cancer (EOC). We treated EOC cells (A2780, HeyA8, RMG1, and HeyA8-MDR) with axitinib to evaluate its effects on cell viabilty, apoptosis and migration. Western blots were performed to assess VEGFR2, ERK, and AKT levels, and ELISA and FACS to evaluate apoptosis according to axitinib treatment. In addition, in vivo experiments in xenografts using A2780, RMG1, and HeyA8-MDR cell lines were performed. We repeated the experiment with patient-derived xenograft models (PDX) of EOC. Axitinib significantly inhibited cell survival and migration, and increased apoptosis in EOC cells. The expression of VEGFR2 and phosphorylation of AKT and ERK in A2780, RMG1, and HeyA8 were decreased with axitinib treatment in dose-dependent manner, but not in HeyA8-MDR. In in vivo experiments, axitinib significantly decreased tumor weight in xenograft models of drug-sensitive (A2780), and clear cell carcinoma (RMG1) and PDX models for platinum sensitive EOC compared to control, but was not effective in drug-resistant cell line (HeyA8-MDR) or heavily pretreated refractory PDX model. Axitinib showed significant anti-cancer effects in drug-sensitive or clear cell EOC cells via inhibition of VEGFR signals associated with cell proliferation, apoptosis and migration, but not in drug-resistant cells.

KSP siRNA/paclitaxel-loaded PEGylated cationic liposomes for overcoming resistance to KSP inhibitors: Synergistic antitumor effects in drug-resistant ovarian cancer.

Despite the promising anticancer effects of kinesin spindle protein (KSP) inhibition, functional plasticity of kinesins induced resistance against KSP inhibitors in a variety of cancers, leading to clinical failure. Additionally, paclitaxel is a widely used anticancer agent, but drug resistance has limited its use in the recurrent cancers. To overcome resistance against KSP inhibitors, we paired KSP inhibition with microtubule stabilization using KSP siRNA and paclitaxel. To enable temporal co-localization of both drugs in tumor cells in vivo, we exploited PEGylated cationic liposomes carrying both simultaneously. Drug synergism study shows that resistance against KSP inhibition can be suppressed by the action of microtubule-stabilizing paclitaxel, because microtubule stabilization prevents a different kinesin Kif15 from replacing all essential functions of KSP when KSP is inhibited. Our combination therapy showed more effective antiproliferative activity in vitro and in vivo than either paclitaxel or KSP siRNA alone. Ultimately, we could observe significantly improved therapeutic effects in the drug-resistant in vivo models, including cell line and patient-derived xenografts. Taken together, our combination therapy provides a potential anticancer strategy to overcome resistance against KSP inhibitors. Particularly, this strategy also provides an efficient approach to improve the therapeutic effects of paclitaxel in the drug-resistant cancers.

CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer.

OBJECTIVE: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). METHODS: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. RESULTS: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P = 0.035 and P = 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P = 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis. CONCLUSION: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant.

Cytoplasmic expression of EGFR shRNA using a modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR silencing and prolongs antitumor effects.

Unusual activation or overexpression of epidermal growth factor receptor (EGFR) has been found in various cancers, and therefore down-regulation of EGFR expression is recognized as a promising strategy for cancer treatment. For decades, RNAi has emerged as an effective solution to regulate gene overexpression, but transient effects of exogenous siRNA have limited the development of EGFR-targeting siRNA therapeutics. Recently, we developed T7 autogene-based hybrid mRNA/DNA system as a non-viral vector for shRNA production and reported the feasibility of long-term silencing for RFP expression as a concept of proof. To investigate its therapeutic availability in cancer therapy, we here modified the hybrid system to stably express EGFR shRNA and confirmed the antitumor effects. When autocatalytic production of T7pol protein in cytoplasm was combined with pT7-driven cytoplasmic transcription for EGFR shRNA, a single transfection lead to stable EGFR silencing in SKOV3 cells for more than 13 days. Also, liposomal systemic administration at two-week intervals resulted in significant inhibition of tumor growth in both SKOV3-bearing mice and PDX models, contrast to the conventional siRNA approach. Our results show an efficient strategy to overcome the temporary effects of synthetic EGFR siRNA in cancer treatment and ultimately provide a potential candidate as an anticancer drug.

Pharmacogenomic analysis of patient-derived tumor cells in gynecologic cancers.

BACKGROUND: Gynecologic malignancy is one of the leading causes of mortality in female adults worldwide. Comprehensive genomic analysis has revealed a list of molecular aberrations that are essential to tumorigenesis, progression, and metastasis of gynecologic tumors. However, targeting such alterations has frequently led to treatment failures due to underlying genomic complexity and simultaneous activation of various tumor cell survival pathway molecules. A compilation of molecular characterization of tumors with pharmacological drug response is the next step toward clinical application of patient-tailored treatment regimens. RESULTS: Toward this goal, we establish a library of 139 gynecologic tumors including epithelial ovarian cancers (EOCs), cervical, endometrial tumors, and uterine sarcomas that are genomically and/or pharmacologically annotated and explore dynamic pharmacogenomic associations against 37 molecularly targeted drugs. We discover lineage-specific drug sensitivities based on subcategorization of gynecologic tumors and identify TP53 mutation as a molecular determinant that elicits therapeutic response to poly (ADP-Ribose) polymerase (PARP) inhibitor. We further identify transcriptome expression of inhibitor of DNA biding 2 (ID2) as a potential predictive biomarker for treatment response to olaparib. CONCLUSIONS: Together, our results demonstrate the potential utility of rapid drug screening combined with genomic profiling for precision treatment of gynecologic cancers.

PLGA Nanoparticles Codelivering siRNAs against Programmed Cell Death Protein-1 and Its Ligand Gene for Suppression of Colon Tumor Growth.

Tumor-infiltrating T lymphocytes highly express programmed cell death protein-1 (PD-1) that interacts with its ligand, programmed cell death protein ligand-1 (PD-L1) on tumors. PD-1/PD-L1 interactions cause functional exhaustion of effector T cells and impair antitumor immunity, allowing tumors to escape immune surveillance. In addition to such extrinsic interactions, tumors proliferate by transmitting intrinsic PD-L1 signals via the mTOR pathway. Here, we simultaneously silenced PD-1 and PD-L1 expressions on CTLs and colon tumors using PD-1 siRNA/PD-L1 siRNA-loaded PLGA nanoparticles and investigated functional activation of tumor-specific CTLs. When compared to a single PD-1 silencing on CTLs or a single PD-L1 silencing on tumors, cosilencing of PD-1/PD-L1 on CTLs and tumors more efficiently promoted effector functions of tumor-specific CTLs. Moreover, PD-L1-silenced tumors inhibited mTOR signaling and showed an antiproliferative response independent of the adaptive immune response. Ultimately, systemic administration of PD-1 and PD-L1 siRNA via PLGA nanoparticles restored the effector functions of tumor-specific CTLs in MC38 tumor-bearing mice. Compared with antitumor effects of single silencing of PD-1 or PD-L1 alone, cosilencing of PD-1 and PD-L1 showed more significant tumor growth suppression and long-term tumor inhibition in colon cancer. Thus, this study provides an efficient therapeutic strategy for achieving immunotherapy in colon cancer.

Field Investigation of Clay Balls in Full-Depth Asphalt Pavement.

Clay ball is a pavement surface defect which refers to a clump in which clay or dirt is mixed with hot asphalt mixture. Clay ball is typically caused by a combination of aggregate contamination of clay or soil, high aggregate moisture, and low production temperature at the asphalt plant. It usually appears a few weeks or months after paving under traffic load, after being liquefied and knocked from the pavement surface. Clay balls can be the source of potholing, raveling, and other issues such as moisture infiltration and reduced ride quality. This paper presents an investigation of the clay balls on US-31 one winter after construction in Hamilton County, Indiana. In order to understand the pavement condition, their severity was measured using both visual observation and infrared image collection system. In addition, a clay ball amount, its distribution pattern, and cores condition were evaluated. A precipitation effect on clay ball formation was investigated for finding a cause of the clay balls. The investigation found that infrared image collection system was appropriate in detecting the clay balls. The clay balls were elliptic in shape with 2.5 cm to 10 cm in diameter, and the maximum clay ball depth was almost penetrating the entire surface course. It was also found that the asphalt paving on the raining days or right after raining could increase the potential of clay balls. Monitoring of aggregate moisture during construction on or after raining days should be able to reduce the risk of clay balls.

Systemic delivery of Eg5 shRNA-expressing plasmids using PEGylated DC-Chol/DOPE cationic liposome: Long-term silencing and anticancer effects in vivo.

Duration of gene silencing due to the short-term silencing effects induced by exogenous siRNA have limited the therapeutic applications of RNAi and the development of RNAi-based therapeutics. We here generated Eg5 shRNA-expressing plasmids using the inverted terminal repeats (ITRs) sequences to produce Eg5 hairpin RNA under the control of U6 promoter. Using PEGylated DC-Chol/DOPE cationic liposomes, we demonstrated that a single systemic administration of Eg5 shRNA-expressing plasmid/liposome lipoplexes induced the long-term Eg5 silencing in the tumor sites of tumor-bearing mice, and ultimately lead to more sustained anticancer effects than standard synthetic siEg5/liposome lipoplexes. This non-viral Eg5 shRNA expression system had no risk of immunogenicity anticipated in the use of viral vectors, and could reduce the potential of off-target effects by scaling down the administration dose of RNAi therapeutics in patient. Therefore, the sustainable shRNA expression properties in the tumor sites suggest an efficient strategy to overcome the limitations caused by chemically synthesized siRNA methods such as short-term silencing effects and off-target effects. Herein, this study provides a non-viral silencing strategy for inducing long-term Eg5 silencing in vivo and suggests the great potential of Eg5 shRNA-expressing lipoplexes as a DNA-based RNAi therapeutics for cancer treatment.

Anti-EpCAM-conjugated adeno-associated virus serotype 2 for systemic delivery of EGFR shRNA: Its retargeting and antitumor effects on OVCAR3 ovarian cancer in vivo.

Adeno-associated virus (AAV) is a promising vector for systemic delivery of siRNA because of its long-term expression ability without immunogenicity and pathogenicity. However, its broad host tropism and lack of tissue specificity have limited clinical applications such as cancer therapy. Therefore, redirecting the natural tropism of AAV vectors to unique cell surface antigens is an important requirement for in vivo RNAi-based cancer therapy. To use the overexpression property of epithelial cell adhesion molecule (EpCAM) in specific cancer types, we herein created anti-EpCAM antibody-conjugated AAV serotype 2 (AAV2) vectors through a streptavidin-biotin bridge. Upon intravenous injection, anti-EpCAM-conjugated AAV2 vectors showed prominent tumor-specific accumulation in EpCAM-positive tumor-bearing mice without undesirable sequestration in liver. In addition, when loaded with transgenes to express shRNA against epidermal growth factor receptor (EGFR), systemically injected anti-EpCAM-conjugated AAV2/shEGFR vectors induced significant downregulation of EGFR expression in tumors and eventually suppressed tumor growth even at the long dosing interval of two weeks. This in vivo antitumor effect represents the increased infection efficacy of tropism-modified AAV2 vectors and prolonged expression of EGFR shRNA in tumor tissues. Thus, this study suggests the great potential of anti-EpCAM-conjugated AAV2/shEGFR vectors as RNAi-based cancer therapeutics. STATEMENT OF SIGNIFICANCE: Adeno-associated virus (AAV) is a promising vector for systemic delivery of siRNA, but its broad host tropism has limited clinical applications. By using the overexpression property of epithelial cell adhesion molecule (EpCAM) on tumors, we demonstrate that anti-EpCAM-conjugated AAV2 vectors through a streptavidin-biotin bridge are redirected to EpCAM-positive tumors in vivo. In addition, when loaded with transgenes to express shRNA against epidermal growth factor receptor (EGFR), systemically injected anti-EpCAM-conjugated AAV2/shEGFR vectors significantly downregulate EGFR expression in tumors, eventually suppressing tumor growth for long periods. We herein suggest the potential of anti-EpCAM-AAV2/shEGFR vectors as an antitumor agent. Furthermore, redirection of AAV2 infection through EpCAM would provide a powerful means for systemic delivery of short hairpin RNA to tumor sites.

A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry.

The transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.

Anatomy of the Superficial Venous Structures of the Neck: A Cadaveric Study to Guide Superficial Injections.

BACKGROUND: Minimally invasive aesthetic procedures of the neck are becoming more popular. However, anatomical studies on the venous structures of the neck in relation to these procedures are lacking. OBJECTIVE: The aims of this study were to identify the locations and communication patterns of the anterior jugular vein and external jugular vein (AJV and EJV) and the communicating vein (CV) based on superficial anatomical landmarks and to determine dangerous areas for dermal filler injections into the neck. MATERIALS AND METHODS: Thirty sides of the neck from Korean adult cadavers were dissected for this study. RESULTS: Four anatomical variants were identified. In Type Ia, the CV ran along the anterior border of the sternocleidomastoid muscle (SCM) (33.4%); in Type Ib, a single vein was observed connecting the CV and the EJV at the level of laryngeal prominence (23.3%); in Type Ic, the CV proceeded separately from the medial side of the anterior border of the SCM (13.3%); and in Type II, the CV was absent while the EJV and AJV were observed (30%). CONCLUSION: Given the 4 anatomical variants identified in this study, the authors recommend exerting caution when performing dermal filler injections approximately 10, 30, and 60 mm lateral to the midsagittal line to avoid iatrogenic side effects.

CD44-Targeting PLGA Nanoparticles Incorporating Paclitaxel and FAK siRNA Overcome Chemoresistance in Epithelial Ovarian Cancer.

Chemotherapy is commonly used in the treatment of ovarian cancer, yet most ovarian cancers harbor inherent resistance or develop acquired resistance. Therefore, novel therapeutic approaches to overcome chemoresistance are required. In this study, we developed a hyaluronic acid-labeled poly(d,l-lactide-co-glycolide) nanoparticle (HA-PLGA-NP) encapsulating both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA as a selective delivery system against chemoresistant ovarian cancer. The mean size and zeta potential of the HA-PLGA-NP were 220 nm and -7.3 mV, respectively. Incorporation efficiencies for PTX and FAK siRNA in the HA-PLGA-NPs were 77% and 85%, respectively. HA-PLGA-NP showed higher binding efficiency for CD44-positive tumor cells as compared with CD44-negative cells. HA-PLGA (PTX+FAK siRNA)-NP caused increased cytotoxicity and apoptosis in drug-resistant tumor cells. Treatment of human epithelial ovarian cancer tumor models HeyA8-MDR (P < 0.001) and SKOV3-TR (P < 0.001) with HA-PLGA (PTX+FAK siRNA)-NP resulted in significant inhibition of tumor growth. Moreover, in a drug-resistant, patient-derived xenograft (PDX) model, HA-PLGA (PTX+FAK siRNA)-NP significantly inhibited tumor growth compared with PTX alone (P < 0.002). Taken together, HA-PLGA-NP acts as an effective and selective delivery system for both the chemotherapeutic and the siRNA in order to overcome chemoresistance in ovarian carcinoma.Significance: These findings demonstrate the efficacy of a novel, selective, two-in-one delivery system to overcome chemoresistance in epithelial ovarian cancer. Cancer Res; 78(21); 6247-56. ©2018 AACR.

PEGylated DC-Chol/DOPE cationic liposomes containing KSP siRNA as a systemic siRNA delivery Carrier for ovarian cancer therapy.

Although siRNA-mediated downregulation technology has been highly successful in suppressing the expression of any disease-related gene, systemic delivery of siRNA for the clinical applications remains challenging, especially in the use of cancer therapy. DC-Chol/DOPE cationic liposomes as one of the most attractive vehicles for gene delivery have been widely exploited for transfection of siRNA into cells, but complexity of systemic delivery has allowed only their direct injection into local targets due to the formation of aggregations with negatively-charged blood components. Herein, we demonstrate the effects of PEGylation on DC-Chol/DOPE cationic liposomes for systemic siRNA delivery in cancer therapy. In contrast to non-PEGylated DC-Chol/DOPE-siRNA lipoplexes, PEGylated DC-Chol/DOPE-siRNA lipoplexes reduce the excretion by kidneys and scavenging in liver, prolonging the circulation time in vivo, and ultimately increase their preferential tumor accumulation. Therefore, systemic injection of PEGylated DC-Chol/DOPE liposomes loaded with siRNA against kinesin spindle protein (KSP) gene exhibited a high level of target gene silencing at tumor sites and substantial suppression of tumor growth. Furthermore, systemically administered PEGylated lipoplexes did not lead to any activation of innate immune responses in the immunocompetent mice. These results suggest the potential of PEGylated DC-Chol/DOPE liposomes as a systemic delivery carrier for siRNA-mediated cancer therapy.

Selective delivery of PLXDC1 small interfering RNA to endothelial cells for anti-angiogenesis tumor therapy using CD44-targeted chitosan nanoparticles for epithelial ovarian cancer.

Angiogenesis plays an essential role in the growth and metastasis of tumor cells, and the modulation of angiogenesis can be an effective approach for cancer therapy. We focused on silencing the angiogenic gene PLXDC1 as an important factor for anti-angiogenesis tumor therapy. Herein, we developed PLXDC1 small interfering siRNA (siRNA)-incorporated chitosan nanoparticle (CH-NP/siRNA) coated with hyaluronic acid (HA) to target the CD44 receptor on tumor endothelial cells. This study aimed to improve targeted delivery and enhance therapeutic efficacy for tumor anti-angiogenesis. The HA-CH-NP/siRNA was 200 ± 10 nm in size with a zeta potential of 26.4 mV. The loading efficiency of siRNA to the HA-CH-NP/siRNA was up to 60%. The selective binding of HA-CH-NP/siRNA to CD44-positive tumor endothelial cells increased by 2.1-fold compared with that of the CD44 nontargeted CH-NP/siRNA. PLXDC1 silencing by the HA-CH-NP/siRNA significantly inhibited tumor growth in A2780 tumor-bearing mice compared with that in the control group (p < .01), and mRNA expression of PLXDC1 was significantly reduced in the HA-CH-NP/siRNA-treated group. Furthermore, treatment with HA-CH-NP/siRNA resulted in significant inhibition of cell proliferation (p < .001), reduced microvessel density (p < .001), and increased cell apoptosis (p < .001). This study demonstrates that HA-CH-NP/siRNA is a highly selective delivery platform for siRNA, and has broad potential to be used in anti-angiogenesis tumor therapy.

PLGA nanoparticles co-delivering MDR1 and BCL2 siRNA for overcoming resistance of paclitaxel and cisplatin in recurrent or advanced ovarian cancer.

The inherent or acquired resistance to paclitaxel and cisplatin, which are commonly used chemotherapeutic agents for ovarian cancer treatment, remains an important issue in chemotherapy of multidrug resistant ovarian cancer. Currently, it is still challenging to deal with the recurrent or advanced stage ovarian cancer. When drug efflux and anti-apoptotic pathways are highly interdependent and also involved in developing the resistance of multidrug resistant ovarian cancer, simultaneous inhibition of both pathways represents the potential targets to enhance the efficacy of chemotherapy. Here, we introduce PLGA nanoparticles system as a "dual RNAi delivery system" to contain both MDR1 and BCL2 siRNA, which is designed for simultaneous inhibition of drug efflux and cell death defense pathways. In the present studies, siRNA-loaded PLGA nanoparticles efficiently elicit the simultaneous suppression of both genes, which consequently shows more enhanced drug-sensitivity than sole suppression of drug efflux or anti-apoptosis in the resistant ovarian cancer cells, owing to the interdependence of both pathways. Our siRNA-loaded PLGA nanoparticles for co-delivering MDR1 and BCL2 siRNA provide an efficient combination therapy strategy to overcome the chemoresistance of paclitaxel and cisplatin on the paclitaxel-resistant SKOV3-TR and cisplatin-resistant A2780-CP20 ovarian cancer respectively.

Rolling Circle Transcription for the Self-Assembly of Multimeric RNAi Structures and Its Applications in Nanomedicine.

The enzymatic process of rolling circle transcription (RCT) enables self-assembly of multimeric RNAi structures from a circular DNA template. The self-assembled RNAi structures can be manipulated easily by simple base pairing rules with short DNA fragments for constructing multifunctional nanoparticles in the field of nanomedicine. Here we describe the method to generate multifunctional RNAi nanoparticles applicable in nanomedicine.

The C-terminus of IGFBP-5 suppresses tumor growth by inhibiting angiogenesis.

Insulin-like growth factor-binding protein 5 (IGFBP-5) plays a role in cell growth, differentiation, and apoptosis. In this study, we found that IGFBP5 was markedly downregulated in ovarian cancer tissue. We investigated the functional significance of IGFBP-5 as a tumor suppressor. To determine functional regions of IGFBP-5, truncation mutants were prepared and were studied the effect on tumor growth. Expression of C-terminal region of IGFBP-5 significantly decreased tumor growth in an ovarian cancer xenograft. A peptide derived from the C-terminus of IGFBP-5 (BP5-C) was synthesized to evaluate the minimal amino acid motif that retained anti-tumorigenic activity and its effect on angiogenesis was studied. BP5-C peptide decreased the expression of VEGF-A and MMP-9, phosphorylation of Akt and ERK, and NF-kB activity, and inhibited angiogenesis in in vitro and ex vivo systems. Furthermore, BP5-C peptide significantly decreased tumor weight and angiogenesis in both ovarian cancer orthotopic xenograft and patient-derived xenograft mice. These results suggest that the C-terminus of IGFBP-5 exerts anti-cancer activity by inhibiting angiogenesis via regulation of the Akt/ERK and NF-kB-VEGF/MMP-9 signaling pathway, and might be considered as a novel angiogenesis inhibitor for the treatment of ovarian cancer.

In vivo stepwise immunomodulation using chitosan nanoparticles as a platform nanotechnology for cancer immunotherapy.

Dentritic cell (DC)-based cancer immunotherapy faces challenges in both efficacy and practicality. However, DC-based vaccination requires multiple injections and elaborates ex vivo manipulation, which substantially limits their use. Therefore, we sought to develop a chitosan nanoparticle (CH-NP)-based platform for the next generation of vaccines to bypass the ex vivo manipulation and induce immune responses via active delivery of polyinosinic-polycytidylic acid sodium salt (poly I:C) to target Toll-like receptor 3 (TLR3) in endosomes. We developed CH-NPs encapsulating ovalbumin (OVA) as a model antigen and poly I:C as the adjuvant in an ionic complex. These CH-NPs showed increased in vivo intracellular delivery to the DCs in comparison with controls after injection into tumor-bearing mice, and promoted DC maturation, leading to emergence of antigen-specific cytotoxic CD8+ T cells. Finally, the CH-NPs showed significantly greater antitumor efficacy in EG.7 and TC-1 tumor-bearing mice compared to the control (p < 0.01). Taken together, these data show that the CH-NP platform can be used as an immune response modulatory vaccine for active cancer immunotherapy without ex vivo manipulation, thus resulting in increased anticancer efficacy.