Young and undamaged recombinant albumin alleviates T2DM by improving hepatic glycolysis through EGFR and protecting islet ß cells in mice.

BACKGROUND: Albumin is the most abundant protein in serum and serves as a transporter of free fatty acids (FFA) in blood vessels. In type 2 diabetes mellitus (T2DM) patients, the reduced serum albumin level is a risk factor for T2DM development and progression, although this conclusion is controversial. Moreover, there is no study on the effects and mechanisms of albumin administration to relieve T2DM. We examined whether the administration of young and undamaged recombinant albumin can alleviate T2DM in mice. METHODS: The serum albumin levels and metabolic phenotypes including fasting blood glucose, glucose tolerance tests, and glucose-stimulated insulin secretion were studied in db/db mice or diet-induced obesity mice treated with saline or young, undamaged, and ultrapure rMSA. Apoptosis assays were performed at tissue and cell levels to determine the function of rMSA on islet ß cell protection. Metabolic flux and glucose uptake assays were employed to investigate metabolic changes in saline-treated or rMSA-treated mouse hepatocytes and compared their sensitivity to insulin treatments. RESULTS: In this study, treatment of T2DM mice with young, undamaged, and ultrapure recombinant mouse serum albumin (rMSA) increased their serum albumin levels, which resulted in a reversal of the disease including reduced fasting blood glucose levels, improved glucose tolerance, increased glucose-stimulated insulin secretion, and alleviated islet atrophy. At the cellular level, rMSA improved glucose uptake and glycolysis in hepatocytes. Mechanistically, rMSA reduced the binding between CAV1 and EGFR to increase EGFR activation leading to PI3K-AKT activation. Furthermore, rMSA extracellularly reduced the rate of fatty acid uptake by islet ß-cells, which relieved the accumulation of intracellular ceramide, endoplasmic reticulum stress, and apoptosis. This study provided the first clear demonstration that injections of rMSA can alleviate T2DM in mice. CONCLUSION: Our study demonstrates that increasing serum albumin levels can promote glucose homeostasis and protect islet ß cells, which alleviates T2DM.

Antigen Peptide Transporter 1 (TAP1) Promotes Resistance to MEK Inhibitors in Pancreatic Cancers.

Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors show limited benefit in Kirsten rat sarcoma (KRAS) mutant pancreatic cancer due to drug resistance. To identify mechanisms of resistance to MEK inhibitor (MEKi), we employed a differential expression analysis of MEKi-sensitive versus MEKi-resistant KRAS-mutant pancreatic cancer cell lines. Here, we report that the antigen peptide transporter 1 (TAP1) expression levels of MEKi-resistant cell lines were notably higher than those of MEKi-sensitive cell lines. Suppression of TAP1 significantly sensitized the MEKi-resistant pancreatic ductal adenocarcinoma (PDAC) cells to MEKi and induced higher apoptotic rate in vitro. Moreover, knockdown of TAP1 in MEKi-resistant tumor significantly decreased tumor growth in vivo. Consistently, overexpression of TAP1 in sensitive PDAC cells resulted in increased resistance to MEKi, both in vitro and in vivo. Mechanistic studies demonstrated that TAP1 promoted chemoresistance by enhancing the transport of MEKi out of PDAC cells, leading to reduced intracellular MEKi concentration and attenuated inhibition of KRAS signaling pathways. Moreover, TAP1 expression increased spheroid formation abilities of PDAC cells. These findings suggest that TAP1 could serve as a potential marker for predicting the response of patients to MEKi. Combination of TAP1 suppression and MEKi may provide a novel therapeutic strategy for PDAC treatment.

Suppression of CCT3 Inhibits Tumor Progression by Impairing ATP Production and Cytoplasmic Translation in Lung Adenocarcinoma.

Heat shock proteins are highly expressed in various cancers and exert critical functions in tumor progression. However, their expression patterns and functions in lung adenocarcinoma (LUAD) remain largely unknown. We identified that chaperonin-containing T-complex protein-1 subunit 3 (CCT3) was highly expressed in LUAD cells and was positively correlated with LUAD malignancy in the clinical samples. Animal studies showed that silencing CCT3 dramatically inhibited tumor growth and metastasis of LUAD. Proliferation and migration were markedly suppressed in CCT3-deficient LUAD cells. Moreover, the knockdown of CCT3 promoted apoptosis and cell cycle arrest. Mechanistically, the function of glycolysis was significantly inhibited and the total intracellular ATP levels were reduced by at least 25% in CCT3-deficient cells. In addition, the knockdown of CCT3 decreased the protein translation and led to a significant reduction in eukaryotic translation initiation factor 3 (EIF3G) protein, which was identified as a protein that interacts with CCT3. Impaired protein synthesis and cell growth in EIF3G-deficient cells were consistent with those caused by CCT3 knockdown in LUAD cells. Taken together, our study demonstrated in multiple ways that CCT3 is a critical factor for supporting growth and metastasis of LUAD, and for the first time, its roles in maintaining intracellular ATP levels and cytoplasmic translation are reported. Our novel findings provide a potential therapeutic target for lung adenocarcinoma.

Extracellular Hsp90α and clusterin synergistically promote breast cancer epithelial-to-mesenchymal transition and metastasis via LRP1.

Extracellular heat shock protein 90 alpha (eHsp90α, also known as HSP90AA1) has been widely reported to promote tumor cell motility and tumor metastasis in various types of cancer. Several extracellular proteins and membrane receptors have been identified as interacting proteins of eHsp90α and mediate its pro-metastasis function. However, the regulatory mechanism of eHsp90α activity remains largely unknown. Here, we report that clusterin, a protein newly demonstrated to interact with eHsp90α, modulates eHsp90α signaling. We found that clusterin potentiated the effects of eHsp90α on activation of the AKT, ERK and NF-κB protein families, epithelial-to-mesenchymal transition (EMT) and migration in breast cancer cells. Furthermore, in vivo investigations demonstrated similar synergistic effects of eHsp90α and clusterin on tumor metastasis. Notably, the effects of eHsp90α and clusterin were mediated by low-density lipoprotein receptor-related protein 1 (LRP1). Proximity ligation assay and co-immunoprecipitation experiments demonstrated that clusterin participated in eHsp90α-LRP1 complex formation, which enhanced the binding affinity of eHsp90α to LRP1. Collectively, our data establish a role of clusterin as a newly discovered modulator of eHsp90α, and unravel detailed molecular mechanisms underlying the synergistic metastasis-promoting effects of clusterin and eHsp90α.

Extracellular Hsp90α Promotes Tumor Lymphangiogenesis and Lymph Node Metastasis in Breast Cancer.

Early detection and discovery of new therapeutic targets are urgently needed to improve the breast cancer treatment outcome. Here we conducted an official clinical trial with cross-validation to corroborate human plasma Hsp90α as a novel breast cancer biomarker. Importantly, similar results were noticed in detecting early-stage breast cancer patients. Additionally, levels of plasma Hsp90α in breast cancer patients were gradually elevated as their clinical stages of regional lymph nodes advanced. In orthotopic breast cancer mouse models, administrating with recombinant Hsp90α protein increased both the primary tumor lymphatic vessel density and sentinel lymph node metastasis by 2 and 10 times, respectively. What is more, Hsp90α neutralizing antibody treatment approximately reduced 70% of lymphatic vessel density and 90% of sentinel lymph node metastasis. In the in vitro study, we demonstrated the role of extracellular Hsp90α (eHsp90α) as a pro-lymphangiogenic factor, which significantly enhanced migration and tube formation abilities of lymphatic endothelial cells (LECs). Mechanistically, eHsp90α signaled to the AKT pathway through low-density lipoprotein receptor-related protein 1 (LRP1) to upregulate the expression and secretion of CXCL8 in the lymphangiogenic process. Collectively, this study proves that plasma Hsp90α serves as an auxiliary diagnosis biomarker and eHsp90α as a molecular mediator promoting lymphangiogenesis in breast cancer.

High expression of CCR5 in melanoma enhances epithelial-mesenchymal transition and metastasis via TGFß1.

Chemokine receptors are highly expressed in various cancers and play crucial roles in tumor progression. However, their expression patterns and functions in melanoma are unclear. The present study aimed to identify the chemokine receptors that play critical roles in melanoma progression and unravel the underlying molecular mechanisms. We found that CCR5 was more abundant in melanoma cells than normal cells and was positively associated with tumor malignancy in clinical patients. Animal experiments suggested that CCR5 deficiency in B16/F10 or A375 cells suppressed primary tumor growth and lung metastasis, whereas CCR5 overexpression in B16/F0 cells enhanced primary tumor growth and lung metastasis. CCR5 played a critical role in proliferation and migration of melanoma cells in vitro. Importantly, CCR5 was required for maintenance of the mesenchymal phenotype of metastatic melanoma cells. Mechanistically, CCR5 positively regulated expression of TGFß1, which in turn induced epithelial-mesenchymal transition and migration via PI3K/AKT/GSK3ß signaling. Collectively, our results establish a critical role of CCR5 expressed by melanoma cells in cancer progression and reveal the novel mechanisms controlling this process, which suggests the prognostic value of CCR5 in melanoma patients and provides novel insights into CCR5-targeted strategies for melanoma treatment. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel pan-cancer biomarker plasma heat shock protein 90alpha and its diagnosis determinants in clinic.

A sensitive and specific diagnosis biomarker, in principle scalable to most cancer types, is needed to reduce the prevalent cancer mortality. Meanwhile, the investigation of diagnosis determinants of a biomarker will facilitate the interpretation of its screening results in clinic. Here we design a large-scale (1558 enrollments), multicenter (multiple hospitals), and cross-validation (two datasets) clinic study to validate plasma Hsp90α quantified by ELISA as a pan-cancer biomarker. ROC curve shows the optimum diagnostic cutoff is 69.19 ng/mL in discriminating various cancer patients from all controls (AUC 0.895, sensitivity 81.33% and specificity 81.65% in test cohort; AUC 0.893, sensitivity 81.72% and specificity 81.03% in validation cohort). Similar results are noted in detecting early-stage cancer patients. Plasma Hsp90α maintains also broad-spectrum for cancer subtypes, especially with 91.78% sensitivity and 91.96% specificity in patients with AFP-limited liver cancer. In addition, we demonstrate levels of plasma Hsp90α are determined by ADAM10 expression, which will affect Hsp90α content in exosomes. Furthermore, Western blotting and PRM-based quantitative proteomics identify that partial false ELISA-negative patients secret high levels of plasma Hsp90α. Mechanism analysis reveal that TGFß-PKCγ gene signature defines a distinct pool of hyperphosphorylated Hsp90α at Theronine residue. In clinic, a mechanistically relevant population of false ELISA-negative patients express also higher levels of PKCγ. In sum, plasma Hsp90α is a novel pan-cancer diagnosis biomarker, and cancer diagnosis with plasma Hsp90α is particularly effective in those patients with high expression of ADAM10, but may be insufficient to detect the patients with low ADAM10 and those with hyperphosphorylated Hsp90α.

Quality, bioactivity study, and preclinical acute toxicity, safety pharmacology evaluation of PEGylated recombinant human endostatin (M2 ES).

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M2 ES was evaluated by silver stain and reversed-phase high-performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M2 ES and endostar. The bioactivity and antitumor efficacy of M2 ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M2 ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M2 ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M2 ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M2 ES and the absence of serious side effect in rhesus monkeys, M2 ES was authorized to initiate a phase I clinical trial.

Biophysical and biological characterization of PEGylated recombinant human endostatin.

Recombinant human endostatin (MES), showing potent inhibition on angiogenesis and tumour growth, has great potential as a therapeutic agent for tumours. The aim of this study was to evaluate the biophysical and biological characterization of PEGylated recombinant human endostatin (M2 ES). Recombinant human endostatin was mono-PEGylated by conjugation with methoxy polyethylene glycol aldehyde (mPEG-ALD), and the modification site was identified by digested peptide mapping and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The purity was assessed by SDS-PAGE, high-performance liquid chromatography (HPLC), and capillary zone electrophoresis. The physicochemical property was analyzed through fluorescence spectroscopy, and circular dichroism. The bioactivity and anti-tumour efficacy of M2 ES were evaluated using an in vitro endothelial cell migration model and a null-mouse xenograft model of a prostatic cancer, respectively. M2 ES molecules contain a single 20 kDa mPEG-ALD molecule conjugated at the N-terminal portion of MES. The purity of M2 ES was greater than 98%. The physicochemical analysis demonstrated that PEGylation does not change the secondary and tertiary structure of MES. Notably, M2 ES retards endothelial cell migration and tumour growth when compared to control group. These biophysical and biological characterization study data contribute to the initiation of the ongoing clinical study.

Endostatin sensitizes p53-deficient non-small-cell lung cancer to genotoxic chemotherapy by targeting DNA-dependent protein kinase catalytic subunit.

Endostatin was discovered as an endogenous angiogenesis inhibitor with broad-spectrum antitumour activities. Although clinical efficacy was observed when endostatin was combined with standard chemotherapy for non-small cell lung cancer (NSCLC), as well as other cancer types, the specific mechanisms underlying the benefit of endostatin are not completely understood. Extensive investigations suggest that endostatin is a multifunctional protein possessing more than anti-angiogenic activity. Here, we found that endostatin exerts a direct chemosensitizing effect on p53-deficient tumour cells. Concomitant treatment with endostatin and genotoxic drugs resulted in therapeutic synergy in both cellular and animal models of p53-deficient NSCLC. Mechanistically, endostatin specifically interacts with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in tumour cells and suppresses its DNA repair activity. Using isogenic NSCLC cells with different p53 statuses, we discovered that p53-deficient tumour cells show chemoresistance to genotoxic drugs, creating a synthetic dependence on DNA-PKcs-mediated DNA repair. In this setting, endostatin exerted inhibitory effects on DNA-PKcs activity, leading to accumulation of DNA lesions and promotion of the therapeutic effect of genotoxic chemotherapy. In contrast, p53-proficient tumour cells were more sensitive to genotoxic drugs so that DNA-PKcs could be cleaved by drug-activated caspase-3, making DNA-PKcs inhibition less effective during this ongoing apoptotic process. Therefore, our data demonstrate a novel mechanism for endostatin as a DNA-PKcs suppressor, and indicate that combination therapy of endostatin with genotoxic drugs could be a promising treatment strategy for cancer patients with p53-deficient tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A pre-clinical safety study of PEGylated recombinant human endostatin (M2ES) in Sprague Dawley rats.

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A pre-clinical study was performed to evaluate the safety of M2ES in rats. After intravenous (IV) infusions of M2ES at a dose level of 3, 15 and 75 mg/kg in Sprague Dawley (SD) rats, M2ES was well tolerated in animals, with no observable changes in clinical observation, body weight, food consumption, urine analysis, hematology and serum biochemical analysis. The increase of kidney weights, and slight to severe vacuolation and necrosis of proximal tubule epithelial cells in kidney were observed in 15 and 75 mg/kg M2ES groups, but this adverse-effect was reversible. In summary, the major toxicity target organ of M2ES might be kidney, and the no observed adverse effect level (NOAEL) of M2ES in rats was 3 mg/kg in this study. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

PLCγ1-PKCγ signaling-mediated Hsp90α plasma membrane translocation facilitates tumor metastasis.

The 90-kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF-mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca(2+) and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell-surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ-induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1-PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.

Specific chemotherapeutic agents induce metastatic behaviour through stromal- and tumour-derived cytokine and angiogenic factor signalling.

Recent studies reveal that chemotherapy can enhance metastasis due to host responses, such as augmented expression of adhesion molecules in endothelial cells and increased populations of myeloid cells. However, it is still unclear how tumour cells contribute to this process. Here, we observed that paclitaxel and carboplatin accelerated lung metastasis in tumour-bearing mice, while doxorubicin and fluorouracil did not. Mechanistically, paclitaxel and carboplatin induced similar changes in cytokine and angiogenic factors. Increased levels of CXCR2, CXCR4, S1P/S1PR1, PlGF and PDGF-BB were identified in the serum or primary tumour tissues of tumour-bearing mice treated by paclitaxel. The serum levels of CXCL1 and PDGF-BB and the tissue level of CXCR4 were also elevated by carboplatin. On the other hand, doxorubicin and fluorouracil did not induce such changes. The chemotherapy-induced cytokine and angiogenic factor changes were also confirmed in gene expression datasets from human patients following chemotherapy treatment. These chemotherapy-enhanced cytokines and angiogenic factors further induced angiogenesis, destabilized vascular integrity, recruited BMDCs to metastatic organs and mediated the proliferation, migration and epithelial-to-mesenchymal transition of tumour cells. Interestingly, inhibitors of these factors counteracted chemotherapy-enhanced metastasis in both tumour-bearing mice and normal mice injected intravenously with B16F10-GFP cells. In particular, blockade of the SDF-1α-CXCR4 or S1P-S1PR1 axes not only compromised chemotherapy-induced metastasis but also prolonged the median survival time by 33.9% and 40.3%, respectively. The current study delineates the mechanism of chemotherapy-induced metastasis and provides novel therapeutic strategies to counterbalance pro-metastatic effects of chemo-drugs via combination treatment with anti-cytokine/anti-angiogenic therapy.

Enhancement of tumor uptake and therapeutic efficacy of EGFR-targeted antibody cetuximab and antibody-drug conjugates by cholesterol sequestration.

Cetuximab, a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), has been intensively investigated as a promising cancer treatment strategy. The specific mechanism of cetuximab endocytosis and its influence on cetuximab uptake, biodistribution and efficacy still remain elusive. Recently, statins have been reported to synergize with EGFR-targeting agents. Our prior work established that nystatin, a cholesterol-sequestering antifungal drug, facilitates endocytosis via the clathrin-dependent pathway. This study aimed to investigate whether nystatin regulates the uptake and efficacy of cetuximab and cetuximab-based antibody-drug conjugates (cetuximab-ADCs). In vitro and in vivo efficacies of nystatin on the uptake and activity of cetuximab/cetuximab-ADCs were studied in multiple human carcinoma cell lines and xenograft models, respectively. We identified that cholesterol sequestration by nystatin enhanced cetuximab internalization in EGFR-positive carcinoma cells by regulating EGFR trafficking/turnover and facilitating a switch from lipid rafts to clathrin-mediated endocytosis. Combination treatment with cetuximab and nystatin selectively increased cetuximab uptake by tumor tissues, translating into potentiated antitumor efficacy of cetuximab in vivo (A431 and A549 tumors). Nystatin-enhanced internalization of cetuximab further improved the uptake and potency of cetuximab-doxorubicin and cetuximab-methotrexate conjugates in EGFR-positive cetuximab-resistant tumors. Combination therapy with nystatin plus either cetuximab or cetuximab-ADC further prolonged animal survival and significantly suppressed tumor growth, as compared with single-agent cetuximab or cetuximab-ADC. In summary, our results identify a novel mechanism whereby cholesterol sequestration enhances the uptake of EGFR-targeting mAb and ADCs, therefore providing preclinical proof-of-concept that combination with nystatin can potentiate the delivery and efficacy of these EGFR-targeted agents.

Endostatin inhibits the tumorigenesis of hemangioendothelioma via downregulation of CXCL1.

Hemangioendotheliomas could be repressed by various anti-angiogenic agents in animal models. It was unclear whether the agents target hemangioendothelioma cells directly. This study elucidated the mechanism by which endostatin inhibited hemangioendothelioma progression. Expression of the endostatin receptors nucleolin and integrin α5ß1 in hemangioendothelioma was assessed by immunohistochemistry. The effects of endostatin on the hemangioendothelioma-derived cells (EOMA) were evaluated by proliferation and apoptosis assays and by angiogenesis array screening. This revealed the contribution of the Chemokine (C-X-C motif) ligand 1 (CXCL1) to hemangioendothelioma progression, which was explored in vitro and in vivo. The clinical relevance of CXCL1 expression in hemangioendothelioma was also evaluated using tissue array. EOMA cells expressed nucleolin and integrin α5ß1 and bound to endostatin. Endostatin did not alter proliferation or hypoxia-induced apoptosis in EOMA cells but it did impair the pro-angiogenic capacity of the cells. Endothelial cell migration was induced by CXCL1 produced by EOMA cells and endostatin downregulated CXCL1 production by inactivating its transcriptional factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In vivo, the knockdown of CXCL1 significantly impaired EOMA cell growth in nude mice; endostatin had no effect when CXCL1 was overexpressed. A strong correlation was observed between CXCL1 levels and hemangioendothelioma occurrence in patients. CXCL1, which was responsible for hemangioendothelioma progression by stimulating angiogenesis, was impaired by endostatin via inactivation of NF-κB in an animal model. In vascular lesions in patients, CXCL1 expression was a negative prognostic factor. CXCL1-inhibting agents such as endostatin may constitute a useful approach to treat the malignant or intermediate vascular lesions.

MicroRNA-542-3p inhibits tumour angiogenesis by targeting angiopoietin-2.

Angiopoietin-2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA-542-3p, which inhibits tumour progression by impairing Ang2's pro-angiogenic activity. In cultured endothelial cells, miR-542-3p inhibited translation of Angpt2 mRNA by binding to its 3' UTR, and addition of miR-542-3p to cultured endothelial cells attenuated angiogenesis. Administration of miR-542-3p to tumour-bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR-542-3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti-angiogenic miR-542-3p directly targets the key angiogenesis-promoting protein Angpt2, suggesting that miR-542-3p may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.

Pharmacokinetics of PEGylated recombinant human endostatin (M2ES) in rats.

AIM: M2ES is PEGylated recombinant human endostatin. In this study we investigated the pharmacokinetics, tissue distribution, and excretion of M2ES in rats. METHODS: (125)I-radiolabeled M2ES was administered to rats by intravenous bolus injection at 3 mg/kg. The pharmacokinetics, tissue distribution and excretion of M2ES were investigated using the trichloroacetic acid (TCA) precipitation method. RESULTS: The serum M2ES concentration-time curve after a single intravenous dose of 3 mg/kg in rats was fitted with a non-compartment model. The pharmacokinetic parameters were evaluated as follows: Cmax=28.3 µg·equ/mL, t1/2=71.5 h, AUC(0-∞)=174.6 µg·equ·h/mL, Cl=17.2 mL·h(-1)·kg(-1), MRT=57.6 h, and Vss=989.8 mL/kg for the total radioactivity; Cmax=30.3 µg·equ/mL, t1/2=60.1 h, AUC(0-∞)=146.2 µg·equ·h/mL, Cl=20.6 mL·h(-1)·kg(-1), MRT=47.4 h, and Vss=974.6 mL/kg for the TCA precipitate radioactivity. M2ES was rapidly and widely distributed in various tissues and showed substantial deposition in kidney, adrenal gland, lung, spleen, bladder and liver. The radioactivity recovered in the urine and feces by 432 h post-dose was 71.3% and 8.3%, respectively. Only 0.98% of radioactivity was excreted in the bile by 24 h post-dose. CONCLUSION: PEG modification substantially prolongs the circulation time of recombinant human endostatin and effectively improves its pharmacokinetic behavior. M2ES is extensively distributed in most tissues of rats, including kidney, adrenal gland, lung, spleen, bladder and liver. Urinary excretion was the major elimination route for M2ES.

The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.

It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr(115)/Thr(425)/Thr(603), of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a 'phosphorylation switch' for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

Pre-clinical toxicokinetics and safety study of M2ES, a PEGylated recombinant human endostatin, in rhesus monkeys.

PEGylated recombinant human endostatin (M2ES) exhibited prolonged serum half-life and enhanced antitumor activity when compared with endostatin. A non-clinical study was performed to evaluate the toxicokinetics and safety of M2ES in rhesus monkeys. After intravenous (IV) infusions of M2ES at a dose level of 3, 10, and 30mg/kg in rhesus monkeys, the concentration-time curves of M2ES were best fitted to a non-compartment model, and area under the curve (AUC) was positively correlated with the dosage. M2ES had a tendency to accumulate in vivo following successive IV infusions. Serum anti-M2ES IgG antibodies were generated quickly during IV administration, and the antibody level in serum did not significantly decrease after four-week recovery period. Animals administered IV infusions twice weekly (M2ES at 10 or 30mg/kg body weight per day) for 3months developed mild or moderate vacuolation of proximal tubule epithelial cell in proximal convoluted tubule of kidney, but this adverse-effect was reversible. In summary, M2ES was well tolerated and did not cause any serious toxicity. These pre-clinical safety data contribute to the initiation of the ongoing clinical study.

Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways.

Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.