Zhuo (Georgia) Chen

Therapeutic Nanoparticles for Drug Delivery in Cancer

Cancer nanotherapeutics are rapidly progressing and are being implemented to solve several limitations of conventional drug delivery systems such as nonspecific biodistribution and targeting, lack of water solubility, poor oral bioavailability, and low therapeutic indices. To improve the biodistribution of cancer drugs, nanoparticles have been designed for optimal size and surface characteristics to increase their circulation time in the bloodstream. They are also able to carry their loaded active drugs to cancer cells by selectively using the unique pathophysiology of tumors, such as their enhanced permeability and retention effect and the tumor microenvironment. In addition to this passive targeting mechanism, active targeting strategies using ligands or antibodies directed against selected tumor targets amplify the specificity of these therapeutic nanoparticles. Drug resistance, another obstacle that impedes the efficacy of both molecularly targeted and conventional chemotherapeutic agents, might also be overcome, or at least reduced, using nanoparticles. Nanoparticles have the ability to accumulate in cells without being recognized by P-glycoprotein, one of the main mediators of multidrug resistance, resulting in the increased intracellular concentration of drugs. Multifunctional and multiplex nanoparticles are now being actively investigated and are on the horizon as the next generation of nanoparticles, facilitating personalized and tailored cancer treatment.

Expression Pattern of Chemokine Receptor 6 (CCR6) and CCR7 in Squamous Cell Carcinoma of the Head and Neck Identifies a Novel Metastatic Phenotype

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes predictably to cervical lymph nodes, with low rates of distant metastases. Tumor cells can express various receptors that facilitate such metastatic spread to lymph nodes and other nonlymphoid organs. Chemokine receptors (CCR), normally expressed on lymphocytes, control immune and inflammatory cell migration, providing a link between innate and adaptive immunity. Chemokine receptor expression was evaluated in SCCHN, using paired primary and metastatic tumors cell lines, and paired primary and metastatic biopsies from the same patients. Quantitative reverse transcription-PCR showed a consistent pattern of CCR6 down-regulation and up-regulation of CCR7 in metastatic cells and tissues. Chemotaxis assays, ligand-induced receptor down-regulation, and specific antibody blocking experiments supported the quantitative reverse transcription-PCR results, indicating that these surface receptors were functional on metastatic tumor cells. Cells derived from a highly metastatic mouse model of SCCHN were used to confirm CCR7 up-regulation in tumor cells with higher metastatic potential. CCR6 down-regulation is consistent with its decreased expression in cells emigrating from peripheral mucosal sites, whereas CCR7, important for homing of immune cells to secondary lymphoid organs, was significantly up-regulated. Thus, CCR6, CCR7, and their ligands, normally important in controlling immune cell trafficking in response to inflammatory stimuli, may have an important role in determining the metastasis of SCCHN cells in vivo.

HFT-T, a Targeting Nanoparticle, Enhances Specific Delivery of Paclitaxel to Folate Receptor-Positive Tumors

Nonspecific distribution of chemotherapeutic drugs (such as paclitaxel) is a major factor contributing to side effects and poor clinical outcomes in the treatment of human head and neck cancer. To develop novel drug delivery systems with enhanced efficacy and minimized adverse effects, we synthesized a ternary conjugate heparin-folic acid-paclitaxel (HFT), loaded with additional paclitaxel (T). The resulting nanoparticle, HFT-T, is expected to retain the antitumor activity of paclitaxel and specifically target folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of paclitaxel. In vitro experiments found that HFT-T selectively recognizes FR-positive human head and neck cancer cell line KB-3-1, displaying higher cytotoxicity compared to the free form of paclitaxel. In a subcutaneous KB-3-1 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably improved antitumor efficacy of paclitaxel. The average tumor volume in the HFT-T treatment group was 92.9 +/- 78.2 mm(3) vs 1670.3 +/- 286.1 mm(3) in the mice treated with free paclitaxel. Furthermore, paclitaxel tumors showed a resurgence of growth after several weeks of treatment, but this was not observed with HFT-T. This indicates that HFT-T could be more effective in preventing tumors from developing drug resistance. No significant acute in vivo toxicity was observed. These results indicate that specific delivery of paclitaxel with a ternary structured nanoparticle (HFT-T) targeting FR-positive tumor is a promising strategy to enhance chemotherapy efficacy and minimize adverse effects.

Simultaneously Targeting Epidermal Growth Factor Receptor Tyrosine Kinase and Cyclooxygenase-2, an Efficient Approach to Inhibition of Squamous Cell Carcinoma of the Head and Neck

Purpose: Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (Cox-2) contribute to development of squamous cell carcinoma of the head and neck (SCCHN). Simultaneously blocking both EGFR and Cox-2–mediated pathways may be an efficient means of inhibiting cancer cell growth in SCCHN. Experimental Design: A combination of EGFR-selective tyrosine kinase inhibitors (TKIs) AG1478 or ZD1839 (Iressa or gefitinib) with a Cox-2 inhibitor (Cox-2I) celecoxib (Celebrex) was studied for its effects on cell growth, cell cycle progression, and apoptosis in SCCHN cell lines by cell growth assay, clonogenic assay, flow cytometric analysis, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. A potential effect of EGFR TKIs and Cox-2I on angiogenesis was examined by endothelial capillary tube formation assay. Primary and secondary targets of EGFR TKIs and Cox-2I were also examined using immunoblotting and immunoprecipitation after the combined treatment. Results: The combination of AG1478 or ZD1839 with celecoxib either additively or synergistically inhibited growth of the five SCCHN cell lines examined, significantly induced G1 arrest and apoptosis, and suppressed capillary formation of endothelium. Furthermore, the combination showed strong reductions of p-EGFR, p-extracellular signal-regulated kinase 1/2, and p-Akt in SCCHN cells as compared with the single agents. Both AG1478 and ZD1839 inhibited expression of Cox-2 protein, whereas celecoxib mainly blocked the production of prostaglandin E2. Conclusions: These results suggest that cell growth inhibition induced by a combination of EGFR TKIs and Cox-2I is mediated through simultaneously blocking EGFR and Cox-2 pathways. This combination holds a great potential for the treatment and/or prevention of SCCHN.

Tumor Growth Inhibition by Simultaneously Blocking Epidermal Growth Factor Receptor and Cyclooxygenase-2 in a Xenograft Model

Purpose: Our previous study revealed that simultaneously targeting epidermal growth factor receptor (EGFR) tyrosine kinase and cyclooxygenase-2 (COX-2) additively or synergistically inhibited growth of squamous cell carcinoma of the head and neck (SCCHN) in vitro. However, an in vivo efficacy of this combined treatment in SCCHN has not been studied. Experimental Design: Nude mice were pretreated with control (1% Tween 80), ZD1839 (50 mg/kg) alone, celecoxib (50 mg/kg) alone, or a combination of ZD1839 and celecoxib at the same dosages for 7 days before injection of a human SCCHN cell line Tu212. The animals were continuously treated with the agents 5 days a week for about 11 weeks. Results: Tumor growth in the combined treatment was significantly inhibited compared with the control (P < 0.001), ZD1839 (P = 0.005), or celecoxib (P < 0.001). At the same time, a dramatic delay of tumor progression was observed in the combined treatment compared with all other three groups. Molecular analysis showed that the combined treatment significantly decreased prostaglandin E metabolite production. The cooperative effect of these two agents in combination was also associated with down-regulation of phosphorylated EGFR, phosphorylated extracellular signal-regulated kinase, and phosphorylated signal transducers and activators of transcription 3 levels and reduction of vascular endothelial growth factor and Ki-67 expression. Specifically, gene silencing of both EGFR and COX-2 by small interfering RNA further confirmed the cooperative antitumor effect. Conclusion: The current results strongly suggest that a cooperative effect of the combined treatment on tumor progression is mediated through blocking both EGFR- and COX-2-related pathways. This combination regimen may provide a promising strategy for cancer therapy and chemoprevention in SCCHN.

CXC Chemokine Receptor-4 Antagonist Blocks Both Growth of Primary Tumor and Metastasis of Head and Neck Cancer in Xenograft Mouse Models

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.

p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and frequently metastasizes to LNs. Identifying metastasis-promoting factors is of immense clinical interest, as the prognosis for patients with even a single unilateral LN metastasis is extremely poor. Here, we report that p90 ribosomal S6 kinase 2 (RSK2) promotes human HNSCC cell invasion and metastasis. We determined that RSK2 was overexpressed and activated in highly invasive HNSCC cell lines compared with poorly invasive cell lines. Expression of RSK2 also correlated with metastatic progression in patients with HNSCC. Ectopic expression of RSK2 substantially enhanced the invasive capacity of HNSCC cells, while inhibition of RSK2 activity led to marked attenuation of invasion in vitro. Additionally, shRNA knockdown of RSK2 substantially reduced the invasive and metastatic potential of HNSCC cells in vitro and in vivo in a xenograft mouse model, respectively. Mechanistically, we determined that cAMP-responsive element-binding protein (CREB) and Hsp27 are phosphorylated and activated by RSK2 and are important for the RSK2-mediated invasive ability of HNSCC cells. Our findings suggest that RSK2 is involved in the prometastatic programming of HNSCC cells, through phosphorylation of proteins in a putative signaling network. Moreover, targeting RSK2 markedly attenuates in vitro invasion and in vivo metastasis of HNSCC cells, suggesting that RSK2 may represent a therapeutic target in the treatment of metastatic HNSCC.

A folate receptor-targeting nanoparticle minimizes drug resistance in a human cancer model.

Resistance to chemotherapy is a major obstacle in cancer therapy. The main purpose of this study is to evaluate the potential of a folate receptor-targeting nanoparticle to overcome/minimize drug resistance and to explore the underlying mechanisms. This is accomplished with enhanced cellular accumulation and retention of paclitaxel (one of the most effective anticancer drugs in use today and a well-known P-glycoprotein (P-gp) substrate) in a P-gp-overexpressing cancer model. The folate receptor-targeted nanoparticle, HFT-T, consists of a heparin-folate-paclitaxel (HFT) backbone with an additional paclitaxel (T) loaded in its hydrophobic core. In vitro analyses demonstrated that the HFT-T nanoparticle was superior to free paclitaxel or nontargeted nanoparticle (HT-T) in inhibiting proliferation of P-gp-overexpressing cancer cells (KB-8-5), partially due to its enhanced uptake and prolonged intracellular retention. In a subcutaneous KB-8-5 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably prolonged retention within tumor tissues. Importantly, HFT-T treatment markedly retarded tumor growth in a xenograft model of resistant human squamous cancer. Immunohistochemical analysis further indicated that increased in vivo efficacy of HFT-T nanoparticles was associated with a higher degree of microtubule stabilization, mitotic arrest, antiangiogenic activity, and inhibition of cell proliferation. These findings suggest that when the paclitaxel was delivered as an HFT-T nanoparticle, the drug is better retained within the P-gp-overexpressing cells than the free form of paclitaxel. These results indicated that the targeted HFT-T nanoparticle may be promising in minimizing P-gp related drug resistance and enhancing therapeutic efficacy compared with the free form of paclitaxel.

Targeted Delivery of Cisplatin to Lung Cancer Using ScFvEGFR- Heparin-Cisplatin Nanoparticles

The clinical application of cis-diamminedichloroplatinum(II) (DDP, cisplatin) for cancer therapy is limited by its nonspecific biodistribution and severe side effects. Here, we have developed EGFR-targeted heparin-DDP (EHDDP) nanoparticles for tumor-targeted delivery of DDP. This nanoparticle delivery system possesses the following unique properties: (i) succinic anhydride-modified heparin is biocompatible and biodegradable with no anticoagulant activity; (ii) single-chain variable fragment anti-EGFR antibody (ScFvEGFR) was conjugated to the nanoparticles as an EGFR-targeting ligand. Our results showed that EHDDP nanoparticles can significantly increase the intracellular concentrations of DDP and Pt-DNA adducts in EGFR-expressing non-small cell lung cancer H292 cells via an EGFR-mediated pathway. Compared to the free DDP, significantly prolonged blood circulation time and improved pharmacokinetics and biodistribution of Pt were observed after systemic delivery of the EHDDP nanoparticles. The new EHDDP nanoparticle delivery system significantly enhanced antitumor activity of DDP without weight loss or damage to the kidney and spleen in nude mice bearing H292 cell tumors.

Synergistic inhibition of head and neck tumor growth by green tea (−)-epigallocatechin-3-gallate and EGFR tyrosine kinase inhibitor

One of the mechanisms of the antitumor activity of green tea (−)‐epigallocatechin‐3‐gallate (EGCG) is associated with its effect on epidermal growth factor receptor (EGFR)‐mediated signaling transduction pathways. We investigated whether combining EGCG with the EGFR‐tyrosine kinase inhibitor (EGFR‐TKI) erlotinib may augment erlotinib‐induced cell growth inhibition of squamous cell carcinoma of the head and neck (SCCHN) in a mouse xenograft model. In vitro studies with 5 head and neck cancer cell lines revealed that synergistic cell growth inhibition by the combination of EGCG and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, increased activation of caspases 9, 3 and PARP compared to the inhibition induced by EGCG or erlotinib alone. Erlotinib inhibited phosphorylation of EGFR, stabilizing EGFR at the plasma membrane, whereas EGCG induced EGFR internalization and ubiquitin‐degradation, ultimately undermining EGFR signaling. The efficacy of the combination treatment was investigated with nude mice (n = 25) orally gavaged with vehicle control, EGCG, erlotinib or the combination at the same doses for 7 days, followed by subcutaneous injection with Tu212 cells. Animals were continuously administered the agents 5 days weekly for 7 weeks. The combined treatment resulted in significantly greater inhibition of tumor growth and delayed tumor progression as a result of increased apoptosis, decreased cell proliferation and reduced pEGFR and pAKT compared to the single agent treatment groups. Our results suggest a synergistic antitumor effect of a combined treatment with EGCG and erlotinib, and provide a promising regimen for future chemoprevention and treatment of SCCHN.