Minghai Tang

Liposomal Quercetin Efficiently Suppresses Growth of Solid Tumors in Murine Models

Purpose: Quercetin is a potent chemotherapeutic drug. Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility. To overcome this limitation, this study is aimed to develop liposomal quercetin and investigate its distribution in vivo and antitumor efficacy in vivo and in vitro. Experimental Design: Quercetin was encapsulated in polyethylene glycol 4000 liposomes. Biodistribution of liposomal quercetin i.v. at 50 mg/kg in tumor-bearing mice was detected by high-performance liquid chromatography. Induction of apoptosis by liposomal quercetin in vitro was tested. The antitumor activity of liposomal quercetin was evaluated in the immunocompetent C57BL/6N mice bearing LL/2 Lewis lung cancer and in BALB/c mice bearing CT26 colon adenocarcinoma and H22 hepatoma. Tumor volume and survival time were observed. The mechanisms underlying the antitumor effect of quercetin in vivo was investigated by detecting the microvessel density, apoptosis, and heat shock protein 70 expression in tumor tissues. Results: Liposomal quercetin could be dissolved in i.v. injection and effectively accumulate in tumor tissues. The half-time of liposomal quercetin was 2 hours in plasma. The liposomal quercetin induced apoptosis in vitro and significantly inhibited tumor growth in vivo in a dose-dependent manner. The optimal dose of liposomal quercetin resulted in a 40-day survival rate of 40%. Quantitative real-time PCR showed that liposomal quercetin down-regulated the expression of heat shock protein 70 in tumor tissues. Immunohistochemistry analysis showed that liposomal quercetin inhibited tumor angiogenesis as assessed by CD31 and induced tumor cell apoptosis. Conclusions: Our data indicated that pegylated liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.

Comparative Proteomics Approach to Screening of Potential Diagnostic and Therapeutic Targets for Oral Squamous Cell Carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.

Mechanism of Cancer Cell Adaptation to Metabolic Stress Proteomics Identification of a Novel Thyroid Hormone-mediated Gastric Carcinogenic Signaling Pathway

Gastric cancer is the second most common cancer worldwide and has a poor prognosis. To determine the mechanism of adaptation to metabolic stress in cancer cells, we used gastric cancer as a model system to reveal the potential signaling pathways involved. Two-dimensional polyacrylamide gel electrophoresis coupled with ESI-Q-TOF MS/MS analysis was used to identify differentially expressed proteins between gastric tumor tissues and the corresponding noncancerous tissues. In total, 107 spots with significant alteration (±over 2-fold, p < 0.05) were positively identified by MS/MS analysis. Altered expression of representative proteins was validated by RT-PCR and Western blotting. Cluster analysis of the changed proteins revealed an interesting group of metabolic proteins, which suggested accumulation of triiodothyronine (T3; the major functional component of thyroid hormone) and overexpression of hypoxia-induced factor (HIF) in gastric carcinoma. These observations were further confirmed by electrochemiluminescence immunoassay and immunohistochemistry. T3-induced expression of HIF1-α and vascular endothelial growth factor was further verified using a gastric cancer cell line and in vivo mouse model. Because the early accumulation of HIF1-α was found to be independent of de novo transcription, we also found that the cytosolic cascade phosphatidylinositol 3-kinase/Akt pathway sensitive to T3 stimulus was involved. Furthermore we demonstrated that T3-induced overexpression of HIF1-α was mediated by fumarate accumulation and could be enhanced by fumarate hydratase inactivation but inhibited by 2-oxoglutarate. These results provide evidence for alteration of metabolic proteins and dysfunction of thyroid hormone regulation in gastric tumors, and a novel thyroid hormone-mediated tumorigenic signaling pathway is proposed. Our findings are considered a significant step toward a better understanding of adaptations to metabolic stress in gastric carcinogenesis.

Proteomic profiling identifies aberrant epigenetic modifications induced by hepatitis B virus X protein.

The hepatitis B virus-encoded X (HBx) protein coactivates transcription of a variety of viral and cellular genes and it is believed to play essential roles in viral replication and hepatocarcinogenesis. To examine the pleiotropic effects of HBx protein on host cell protein expression, we utilized 2-DE and MS analysis to compare and identify differentially expressed proteins between a stable HBx-transfected cell line (HepG2-HBx), constitutively expressing HBx, and vector control cells. Of the 60 spots identified as differentially expressed (+/- over 2-fold, p < 0.05) between the two cell lines, 54 spots were positively identified by MS/MS analysis. Several recent studies suggested that HBx was involved in regional hypermethylation of tumor suppressor genes and global hypomethylation of satellite 2 repeats during hepatocarcinogenesis; however, no specific gene has been reported as hypomethylated by HBx. Promoter methylation analysis was examined for those protein spots showing significant alterations, and our results revealed that specific genes, such as aldehyde dehydrogenase 1 (ALDH1), can be hypomethylated by HBx, and two calcium ion-binding proteins, S100A6 and S100A4, were hypermethylated by HBx and could be re-expressed by AZA (DNA methylase inhibitor) treatment. Moreover, via cluster and pathway analysis, we proposed a hypothetical model for the HBx regulatory circuit involving aberrant methylation of retinol metabolism-related genes and calcium homeostasis-related genes. In summary, we profiled proteome alterations between HepG2-HBx and control cells, and found that HBx not only induces regional hypermethylation but also specific hypomethylation of host cell genes.

Comparative proteomics and correlated signaling network of rat hippocampus in the pilocarpine model of temporal lobe epilepsy

In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. The pilocarpine‐induced TLE provides a model to investigate the molecular and functional characterization of epileptogenesis by mimicking the human epileptic condition. Here, we employed a 2‐D gel‐based proteomic technique to profile proteome changes in the rat hippocampus after pilocarpine treatment. Using MALDI MS and MS/MS, 57 differentially expressed proteins were identified, which were found either up‐regulated and/or down‐regulated at the two time points 12 h (acute period; Ap) and 72 h (silent period; Sp) compared with the control. These proteins can be related to underlying mechanism of pilocarpine‐induced TLE, indicating cytoskeleton modification, altered synaptic function, mitochondrial dysfunction, changed ion channel, and chaperone. Five of the identified proteins, synaptosomal‐associated protein 25 (SNAP25), synapsin‐2 (SYN2), homer protein homolog 2 (HOMER2), α‐internexin (INA), and voltage‐dependent anion channel 2 (VDAC2) were investigated by semiquantitative RT‐PCR, and SNAP25 and INA were further validated by Western blot and immunohistochemistry staining. Furthermore, association of these pilocarpine‐induced proteins with biological functions using the Ingenuity Pathway Analysis (IPA) tool showed that nucleic acid metabolism, system development, tissue and cell morphology were significantly altered. IPA of the canonical networks indicated that six membrane proteins (e.g., SNAP25, SYN2, and HOMER2) participated in three biological networks as starting proteins. Our results offer a clue to identify biomarkers for the development of pharmacological therapies targeted at epilepsy.

Comparative Proteomic Approach Identifies Pkm2 and Cofilin-1 as Potential Diagnostic, Prognostic and Therapeutic Targets for Pulmonary Adenocarcinoma

Lung cancer is the leading cause of cancer-related death in the world. Non-small cell lung carcinomas (Non-SCLC) account for almost 80% of lung cancers, of which 40% were adenocarcinomas. For a better understanding of the molecular mechanisms behind the development and progression of lung cancer, particularly lung adenocarcinoma, we have used proteomics technology to search for candidate prognostic and therapeutic targets in pulmonary adenocarcinoma. The protein profile changes between human pulmonary adenocarcinoma tissue and paired surrounding normal tissue were analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE) based approach. Differentially expressed protein-spots were identified with ESI-Q-TOF MS/MS instruments. As a result, thirty two differentially expressed proteins (over 2-fold, p<0.05) were identified in pulmonary adenocarcinoma compared to normal tissues. Among them, two proteins (PKM2 and cofilin-1), significantly up-regulated in adenocarcinoma, were selected for detailed analysis. Immunohistochemical examination indicated that enhanced expression of PKM2 and cofilin-1 were correlated with the severity of epithelial dysplasia, as well as a relatively poor prognosis. Knockdown of PKM2 expression by RNA interference led to a significant suppression of cell growth and induction of apoptosis in pulmonary adenocarcinoma SPC-A1 cells in vitro, and tumor growth inhibition in vivo xenograft model (P<0.05). In addition, the shRNA expressing plasmid targeting cofilin-1 significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. While additional works are needed to elucidate the biological significance and molecular mechanisms of these altered proteins identified in this study, PKM2 and cofilin-1 may serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets for pulmonary adenocarcinoma.

Liposomal honokiol inhibits VEGF-D-induced lymphangiogenesis and metastasis in xenograft tumor model

Lymph nodes metastasis of tumor could be a crucial early step in the metastatic process. Induction of tumor lymphangiogenesis by vascular endothelial growth factor‐D may play an important role in promoting tumor metastasis to regional lymph nodes and these processes can be inhibited by inactivation of the VEGFR‐3 signaling pathway. Honokiol has been reported to possess potent antiangiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor‐associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we established lymph node metastasis models by injecting overexpressing VEGF‐D Lewis lung carcinoma cells into C57BL/6 mice to explore the effect of honokiol on tumor‐associated lymphangiogenesis and related lymph node metastasis. The underlying mechanisms were systematically investigated in vitro and in vivo. In in vivo study, liposomal honokiol significantly inhibited the tumor‐associated lymphangiogenesis and metastasis in Lewis lung carcinoma model. A remarkable delay of tumor growth and prolonged life span were also observed. In in vitro study, honokiol inhibited VEGF‐D‐induced survival, proliferation and tube‐formation of both human umbilical vein endothelial cells (HUVECs) and lymphatic vascular endothelial cells (HLECs). Western blotting analysis showed that liposomal honokiol‐inhibited Akt and MAPK phosphorylation in 2 endothelial cells, and downregulated expressions of VEGFR‐2 of human vascular endothelial cells and VEGFR‐3 of lymphatic endothelial cells. Thus, we identified for the first time that honokiol provided therapeutic benefit not only by direct effects on tumor cells and antiangiogenesis but also by inhibiting lymphangiogenesis and metastasis via the VEGFR‐3 pathway. The present findings may be of importance to investigate the molecular mechanisms underlying the spread of cancer via the lymphatics and explore the therapeutical strategy of honokiol on antilymphangiogenesis and antimetastasis.

Synergistic Antitumor Effects of Liposomal Honokiol Combined with Adriamycin in Breast Cancer Models

Honokiol, a novel antitumor agent, could induce apoptosis and inhibit the growth of vascular endothelium in several tumor cell lines and xenograft models. It has been suggested that the antitumor effect of chemotherapy could be increased by combining it with an antiangiogenesis agent in anticancer strategy. The present study explored the potential to increase the antitumor effect of adriamycin by combining it with honokiol in mouse 4T1 breast cancer models, and the underlining mechanism was investigated. Honokiol was encapsulated in liposomes to improve the water insolubility. In vitro, liposomal honokiol inhibited the proliferation of 4T1 cells via apoptosis and significantly enhanced the apoptosis of 4T1 cells induced by adriamycin. In vivo, the systemic administration of liposomal honokiol and adriamycin significantly decreased tumor growth through increased tumor cell apoptosis compared with either treatment alone. Collectively, these findings suggest that liposomal honokiol may augment the induction of apoptosis in 4T1 cells in vitro and in vivo, and this combined treatment has shown synergistic suppression in tumor progression according to the analysis of isobologram. The present study may be important in future exploration of the potential application of the combined approach in the treatment of breast cancer. Copyright

Liposomal honokiol, a potent anti-angiogenesis agent, in combination with radiotherapy produces a synergistic antitumor efficacy without increasing toxicity

Honokiol is an active compound purified from magnolia that has been shown to induce cell differentiation, apoptosis, and anti-angiogenesis effects, as well as an enhancement in tumor growth delay in combination with chemotherapeutic agents in several mouse xenograft models. Our goal was to investigate the radiosensitization effect of honokiol on lung carcinoma. The radiosensitization effect of liposomal honokiol in Lewis lung carcinoma cells (LL/2) was analyzed using an in vitro clonogenic survival assay. For an in vivo study, Lewis lung carcinoma-bearing C57BL/6 mice were treated with either liposomal honokiol at 25 mg/kg or 5 Gy of single tumor radiation, or a combination of both over 12 days of treatment. The tumor growth delay and the survival time were evaluated. In addition, histological analysis of tumor sections was performed to examine changes by detecting the microvessel density and apoptosis in tumor tissues. In the clonogenic survival assay, LL/2 cells treated with IC50 Lipo-HNK for 24 h showed a radiation enhancement ratio of 1.9. After 12 days of combination treatment, the tumor volume decreased 78% and produced an anti-tumor activity 1.3-fold greater than a predicted additive effect of honokiol and radiation alone. This combination treatment also caused an 8.7 day delay in tumor growth. The cell cycle distribution and histological analysis demonstrated that liposomal honokiol has an anti-tumor effect via inducing apoptosis and inhibiting angiogenesis. Liposomal honokiol can enhance tumor cell radiosensitivity in vitro and in vivo, indicating that radiotherapy combined with liposomal honokiol can lead to greater anti-tumor efficacy.

Synthesis and Biological Evaluation of Novel Millepachine Derivatives As a New Class of Tubulin Polymerization Inhibitors

Twenty-one novel derivatives of millepachine were synthesized and evaluated for their in vitro antiproliferative activity. Among them, 8 exhibited the most potent activity, with IC50 values of 8-27 nM against panel of cancer cell lines and retained full activity in multidrug resistant cancer cells. Treated cells were arrested in G2/M phase and resulted in cellular apoptosis. Microtubule dynamics confirmed 8 was a novel tubulin polymerization inhibitor by binding at the colchicine site. 8 also exhibited antivascular activity because it concentration dependently reduced the cell migration and disrupted capillary like tube formation in HUVEC cells. Furthermore, the hydrochloride salt of 8 (8·HCl) significantly improved the bioavailability up to 47% while retaining the antiproliferative activity. Importantly, 8·HCl significantly inhibited tumor growths in four xenograft models including resistance tumor-cell-bearing mice models without causing significant loss of body weight, suggesting that 8 is a promising new orally anticancer agent to be developed.