α-苦瓜素及α-苦瓜素-PEG体外抗HSV-1的研究

目的研究苦瓜籽核糖体失活蛋白α-苦瓜素(α-MMC)体外抗单纯疱疹病毒(HSV-1)的活性及其聚乙二醇修饰物(α-MMC-PEG)保留这一活性的程度。方法采用MTT法测定α-MMC和α-MMC-PEG对Vero细胞的体外毒性;通过对所培养的上清液中HSV-1囊膜糖蛋白抗原的测定、细胞和病毒的预处理试验,研究了α-MMC和α-MMC-PEG体外抗HSV-1的活性。结果α-MMC和α-MMC-PEG对HSV-1囊膜糖蛋白抗原分泌的抑制作用均呈剂量依赖性,IC50分别为0.67、2.94μmol.L-1;相同剂量下,α-MMC-PEG可保留60%天然α-MMC的抑制活性。3.3μmol.L-1的α-MMC和α-MMC-PEG直接作用病毒后使Vero细胞的存活率分别提高了22%、17%。结论α-MMC经PEG修饰后可保留较高的抗HSV-1活性,修饰前后都对HSV-1有一定的直接灭活作用。     

An α-tocopheryl succinate enzyme-based nanoassembly for cancer imaging and therapy

Nanoassembly (NA) based on a D-α-tocopherol succinate (αTS) conjugated lysozyme (Lys) (Lys-αTS) was fabricated for tumor-selective delivery of curcumin (CUR) for breast cancer therapy. Lys and αTS were used as a biocompatible enzyme and a hydrophobic residue, respectively, for the preparation of nanocarriers in this study. Compared with CUR-loaded cross-linked Lys (c-Lys/CUR) NA, Lys-αTS/CUR NA exhibited a smaller hydrodynamic size (213?nm mean diameter), a narrower size distribution, and a more spherical shape. Sustained drug release was observed from the Lys-αTS/CUR NA for five days at a normal physiological pH (pH 7.4). The developed Lys-αTS/CUR NA showed enhanced cellular accumulation, antiproliferative effects, and apoptotic efficacies in MDA-MB-231 human breast adenocarcinoma cells. According to the results of optical imaging test in the MDA-MB-231 tumor-bearing mouse models, the Lys-αTS/CUR NA-injected group exhibited a more tumor-selective accumulation pattern, rather than being distributed in the normal tissues and organs. The observed tumor targetability of Lys-αTS/CUR was further studied, which revealed improved in vivo anticancer activities (better inhibition of tumor growth and induction of apoptosis in the tumor tissue) after an intravenous administration in the MDA-MB-231 tumor-bearing mouse models. All these results indicate that the newly developed enzyme-based nanocarrier, the Lys-αTS NA, can be a promising candidate for the therapy of breast cancers.     

Evaluation of doxorubicin-loaded pH-sensitive polymeric micelle release from tumor blood vessels and anticancer efficacy using a dorsal skin-fold window chamber model

Aim： To evaluation the doxorubicin （DOX）-loaded pH-sensitive polymeric micelle release from tumor blood vessels into tumor interstitium using an animal vessel visibility model, the so-called dorsal skin-fold window chamber model.
Methods： DOX-loaded pH-sensitive polyHis-b-PEG micelles and DOX-loaded pH-insensitive PLLA-b-PEG micelles were prepared. The uptake of the micelles by MDA-MB-231 breast cancer cells in vitro and in vivo was examined using flow cytometry. The pharmacokinetic parameters of the micelles were determined in SD rats after intravenous injection of a DOX dose （6 mg/kg）. The release of the micelles from tumor vasculature and the antitumor efficacy were evaluated in MDA-MB-231 breast cancer xenografted in nude mice using a dorsal skin-fold window chamber.
Results： The effective elimination half-life t1/2 of the pH-sensitive, pH-insensitive polymeric micelles and DOX-PBS in rats were 11.3 h, 9.4 h, and 2.1 h, respectively. Intravital microscopy in MDA-MB-231 breast cancer xenografted in nude mice showed that the pH-sensitive polymeric micelles rapidly extravasated from the tumor blood vessels, and DOX carried by the pH-sensitive micelles was preferentially released at the tumor site as compared to the pH-insensitive polymeric micelles. Furthermore, the pH-sensitive polymeric micelles exhibited significant greater efficacy in inhibition of tumor growth in the nude mice.
Conclusion： When DOX is loaded into pH-sensitive polymeric micelles, the acidity in tumor interstitium causes the destabilization of the micelles and triggers drug release, resulting in high local concentrations within the tumor, thus more effectively inhibiting the tumor growth in vivo.     

槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤作用研究

目的 探究白屈菜红碱（CHE）对腺样囊性癌细胞（ACC2）生长的抑制作用及机制。方法 利用CCK8法、EdU法、Hoechst33342/PI双染色法、试剂盒法检测CHE对ACC2细胞活力、细胞增殖、细胞凋亡和活性氧（ROS）水平的影响;通过Western blotting技术检测CHE对Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达的影响;利用斑马鱼移植瘤模型检测CHE对斑马鱼体内ACC2细胞生长的抑制作用。结果 CCK-8结果显示：与对照组比较,2、3、4、5、6、7、8、9、10 μmol/L的CHE显著降低ACC2细胞的存活率（P<0.05、0.01）,且呈浓度相关性; ROS检测结果显示：与对照组比较,5、8 μmol/L的CHE导致ACC2细胞内的ROS水平显著上升（P<0.05、0.01）; EdU增殖检测结果表明：与对照组比较,5、8 μmol/L的CHE致使ACC2细胞的增殖能力显著下降（P<0.01）;Hoechst/PI染色结果显示：与对照组比较,CHE 5、8 μmol/L组ACC2细胞凋亡率显著上升（P<0.01）。抗氧化剂N-乙酰半胱氨酸（NAC）显著抑制CHE诱导的ROS水平升高、细胞凋亡增加（P<0.01）;Western blotting结果显示：2、5、8 μmol/L的CHE能够显著上调Cleaved-Caspase 3、PARP、NF-κB蛋白的表达（P<0.01）,且呈现浓度相关性,5、8 μmol/L的CHE能够显著上调p-JNK的蛋白表达（P<0.01）,8 μmol/L的CHE能够显著上调p-p38的蛋白表达（P<0.01）;NAC显著降低由CHE导致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达增加（P<0.01）,5、8 μmol/L CHE能够有效抑制斑马鱼体内肿瘤的生长（P<0.01）。结论 体外及斑马鱼移植瘤模型证明,CHE可以有效抑制ACC2细胞生长,其机制与提高细胞ROS水平,上调NF-κB、p-JNK、p-p38表达,从而抑制细胞增殖、诱导细胞凋亡相关。     

A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo

The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginine-glycine-aspartic acid (cRGD) peptides, which selectively bind to αvβ3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5′-end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an αvβ3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48?h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGD-siEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.     

l-Peptide functionalized dual-responsive nanoparticles for controlled paclitaxel release and enhanced apoptosis in breast cancer cells

Nanoparticles and macromolecular carriers have been widely used to increase the efficacy of chemotherapeutics, largely through passive accumulation provided by their enhanced permeability and retention effect. However, the therapeutic efficacy of nanoscale anticancer drug delivery systems is severely truncated by their low tumor-targetability and inefficient drug release at the target site. Here, the design and development of novel l-peptide functionalized dual-responsive nanoparticles (l-CS-g-PNIPAM-PTX) for active targeting and effective treatment of GRP78-overexpressing human breast cancer in vitro and in vivo are reported. l-CS-g-PNIPAM-PTX NPs have a relative high drug loading (13.5%) and excellent encapsulation efficiency (74.3%) and an average diameter of 275?nm. The release of PTX is slow at pH 7.4 and 25?°C but greatly accelerated at pH 5.0 and 37?°C. MTT assays and confocal experiments showed that the l-CS-g-PNIPAM-PTX NPs possessed high targetability and antitumor activity toward GRP78 overexpressing MDA-MB-231 human breast cancer cells. As expected, l-CS-g-PNIPAM-PTX NPs could effectively treat mice bearing MDA-MB-231 human breast tumor xenografts with little side effects, resulting in complete inhibition of tumor growth and a high survival rate over an experimental period of 60 days. These results indicate that l-peptide-functionalized acid – and thermally activated – PTX prodrug NPs have a great potential for targeted chemotherapy in breast cancer.     

Tannic acid ameliorates doxorubicin-induced cardiotoxicity and potentiates its anti-cancer activity: potential role of tannins in cancer chemotherapy

Doxorubicin, an anthracycline antibiotic, is widely used in the treatment of various solid tumors including breast cancer. However, its use is limited due to a variety of toxicities including cardiotoxicity. The present study aimed to evaluate the effect of tannic acid, a PARG/PARP inhibitor and an antioxidant, on doxorubicin-induced cardiotoxicity in H9c2 embryonic rat heart myoblasts and its anti-cancer activity in MDA-MB-231 human breast cancer cells as well as in DMBA-induced mammary tumor animals. Doxorubicin-induced cardiotoxicity was assessed by measurement of heart weight, plasma LDH level and histopathology. Bcl-2, Bax, PARP-1 and p53 expression were examined by western blotting. Our results show that tannic acid prevents activation of PARP-1, reduces Bax and increases Bcl-2 expression in H9c2 cells, thus, preventing doxorubicin-induced cell death. Further, it reduces the cell viability of MDA-MB-231 breast cancer cells, increases p53 expression in mammary tumors and shows maximum tumor volume reduction, suggesting that tannic acid potentiates the anti-cancer activity of doxorubicin. To the best of our knowledge, this is the first report which shows that tannic acid ameliorates doxorubicin-induced cardiotoxicity and potentiates its anti-cancer activity both in vitro (H9c2 and MDA-MB-231 cells) as well as in in vivo model of DMBA-induced mammary tumor animals.     

Cell penetrating peptides functionalized gambogic acid-nanostructured lipid carrier for cancer treatment

Tumor-targeted delivery is considered a crucial component of current anticancer drug development and is the best approach to increase the efficacy and reduce the toxicity. Nanomedicine, particularly ligand-based nanoparticles have shown a great potential for active targeting of tumor. Cell penetrating peptide is one of the promising ligands in a targeted cancer therapy. In this study, the gambogic acid-loaded nanostructured lipid carrier (GA-NLC) was modified with two kinds of cell penetrating peptides (cRGD and RGERPPR). The GA-NLC was prepared by emulsification and solvent evaporation method and coupled with cRGD, RGERPPR, and combination cRGD and RGERPPR to form GA-NLC-cRGD, GA-NLC-RGE, and GA-NLC-cRGD/RGE, respectively. The formulations were characterized by their particle size and morphology, zeta potential, encapsulation efficiency, and differential scanning calorimetry. In vitro cytotoxicity and cellular uptake study of the formulations were performed against breast cancer cell (MDA-MB-231). Furthermore, in vivo biodistribution and antitumor activity of the formulations were determined by in vivo imaging and in tumor-bearing nude mice, respectively. The result of in vitro cytotoxicity study showed that GA-NLC-RGE exhibited a significantly higher cytotoxicity on MDA-MB-231 as compared with GA-NLC and GA-Sol. Similarly, RGE-Cou-6-NLC showed remarkably higher uptake by the cells than other NLCs over the incubation period. The in vivo imaging study has demonstrated that among the formulations, the RGE-decorated DiR-NLC were more accumulated in the tumor site. The in vivo antitumor activity revealed that RGE-GA-NLC inhibits the tumor growth more efficiently than other formulations. In conclusion, RGERPPR has a potential as an effective carrier in targeting drug delivery of anticancer agents.     

Acetaminophen-induced differentiation of human breast cancer stem cells and inhibition of tumor xenograft growth in mice

It is now believed that cancer stem cells (CSCs) that are resistant to chemotherapy due to their undifferentiated nature drive tumor growth, metastasis and relapse, so development of drugs that induce differentiation of CSCs should have a profound impact on cancer eradication. In this study, we screened medicines that are already in clinical use for drugs that induce differentiation of CSCs. We used MDA-MB-231, a human breast cancer cell line that contains cancer stem cell-like cells. We found that acetaminophen, an anti-inflammatory, antipyretic and analgesic drug, induces differentiation of MDA-MB-231 cells. Differentiation was assessed by observing alterations in cell shape and expression of differentiated and undifferentiated cell markers, a decrease in cell invasion activity and an increase in susceptibility to anti-tumor drugs. This increased susceptibility seems to involve suppression of expression of multidrug efflux pumps. We also suggest that this induction of differentiation is mediated by inhibition of a Wnt/β-catenin canonical signaling pathway. Treatment of MDA-MB-231 cells with acetaminophen in vitro resulted in the loss of their tumorigenic ability in nude mice. Furthermore, administration of acetaminophen inhibited the growth of tumor xenografts of MDA-MB-231 cells in both the presence and absence of simultaneous administration of doxorubicine, a typical anti-tumor drug for breast cancer. Analysis with various acetaminophen derivatives revealed that o-acetamidophenol has a similar differentiation-inducing activity and a similar inhibitory effect on tumor xenograft growth. These results suggest that acetaminophen may be beneficial for breast cancer chemotherapy by inducing the differentiation of CSCs.     

In vivo pharmacokinetics,biodistribution and anti-tumor effect of paclitaxel-loaded targeted chitosan-based polymeric micelle

Abstract

A water-insoluble anti-tumor agent, paclitaxel (PTX) was successfully incorporated into novel-targeted polymeric micelles based on tocopherol succinate-chitosan-polyethylene glycol-folic acid (PTX/TS-CS-PEG-FA). The aim of the present study was to evaluate the pharmacokinetics, tissue distribution and efficacy of PTX/TS-CS-PEG-FA in comparison to Anzatax® in tumor bearing mice. The micellar formulation showed higher in vitro cytotoxicity against mice breast cancer cell line, 4T1, due to the folate receptor-mediated endocytosis. The IC₅₀ value of PTX, a concentration at which 50% cells are killed, was 1.17 and 0.93?µM for Anzatax® and PTX/TS-CS-PEG-FA micelles, respectively. The in vivo anti-tumor efficacy of PTX/TS-CS-PEG-FA, as measured by reduction in tumor volume of 4T1 mouse breast cancer injected in Balb/c mice was significantly greater than that of Anzatax®. Pharmacokinetic study in tumor bearing mice revealed that the micellar formulation prolonged the systemic circulation time of PTX and the AUC of PTX/TS-CS-PEG-FA was obtained 0.83-fold lower than Anzatax®. Compared with Anzatax®, the V_d, T_1/2ß and MRT of PTX/TS-CS-PEG-FA was increased by 2.76, 2.05 and 1.68-fold, respectively. As demonstrated by tissue distribution, the PTX/TS-CS-PEG-FA micelles increased accumulation of PTX in tumor, therefore, resulted in anti-tumor effects enhancement and drug concentration in the normal tissues reduction. Taken together, our evaluations show that PTX/TS-CS-PEG-FA micelle is a potential drug delivery system of PTX for the effective treatment of the tumor and systematic toxicity reduction, thus, the micellar formulation can provide a useful alternative dosage form for intravenous administration of PTX.     

ATP chemosensitivity testing of new antitumor duplex drugs linking 3`-C-ethynylycytidine (ECyd) and 2´-deoxy-5-fluorouridine (5-FdU) in comparison to standard cytostatica and combinations thereof

Background 2′-Deoxy-5-fluorouridylyl-(5′-5′)-3′-C-ethynylcytidine [5-FdU(5′-5′)ECyd] and 3′-C-ethynylcytidinylyl-(5′->1-O)-2-O-octadecyl-sn-glycerylyl-(3-Ο−>5′)-2′-deoxy-5-fluorouridine [ECyd-lipid-5-FdU] are antitumor active duplex drugs and these heterodinucleoside phosphate analogues could be cleaved in vivo by wide-spread phosphodiesterases into different antitumor active antimetabolites. Methods We cultured breast MCF-7, MDA-MB-231 and ovarian OVCAR-29 and OAW-42 cancer cell lines and used the luminometric measuring of the ATP tumor chemosensitivity assay to assess the in vitro activity of 5-FdU(5′-5′)ECyd and ECyd-lipid-5-FdU in comparison to standard single cytostatic agents and combinations thereof currently used in anticancer therapies. To allow comparison between samples and different regimens IndexSUM was determined based on the percentage tumor cell growth inhibition at each test drug concentration. Additionally, the cytostatic efficacy of 5-FdU(5′-5′)ECyd and ECyd-lipid-5-FdU was evaluated at a minimum of five concentrations at 10 fold dilutions using 60 human tumor cell lines including ovarian and breast cancer cell lines from the National Cancer Institute (USA). Results 5-FdU(5′-5′)ECyd and ECyd-lipid-5-FdU have a high cytostatic efficacy reaching 50% tumor cell growth inhibition at concentrations ranging between nano- and micomolar. IndexSum values for broad range efficacy in MCF-7 breast cancer cells were comparable to values obtained for standard drug combinations. Higher cytostatic efficacy was observed in MDA-MB-231 cells. Conclusion The duplex drugs 5-FdU(5′-5′)ECyd and ECyd-lipid-5-FdU represent potential new chemotherapeutic drugs for breast and ovarian cancer cells which are comparable to currently used drug combinations and more potent in comparison to some monocytostatica used in cancer therapy.     

Antitumor Effect of Paclitaxel-Loaded PEGylated Immunoliposomes Against Human Breast Cancer Cells

Purpose The antitumor effect of paclitaxel-loaded PEGylated immunoliposome (PILs) was investigated in breast cancer cell lines and the xenograft model. Methods Herceptin was conjugated to paclitaxel-loaded PEGylated liposomes (PLs). In vitro cellular uptake and cytotoxicity of PILs were determined in breast cancer cell lines while in vivo antitumor efficacy was evaluated in the xenograft nude mouse model. Results The PILs formulation was able to significantly increase the HER2 mediated cellular uptake of paclitaxel compared to the PLs in cell lines overexpressing HER2 (BT-474 and SK-BR-3 cells). However, in the MDA-MB-231 cells, which express low levels of HER2, the difference between the PILs and PLs formulation was not significant. The biological activity of Herceptin was maintained throughout the conjugation process as exhibited by the antitumor dose–response curves determined for Herceptin itself, for the thiolated Herceptin alone and subsequently for the immunoliposome-coupled Herceptin. In BT-474 and SK-BR-3 cells, the cytotoxicity of the PILs was more potent than that of Taxol. Moreover, in in vivo studies, PILs showed significantly higher tumor tissue distribution of paclitaxel in the BT-474 xenograft model and more superior antitumor efficacy compared to Taxol and PLs. However, in the MDA-MB-231 xenograft model, PILs and PLs showed similar tumor tissue distribution as well as antitumor activity. Conclusions These results suggest that HER2-mediated endocytosis is involved in the PILs formulation. The ability of the PILs formulation to efficiently and specifically deliver paclitaxel to the HER2-overexpressing cancer cells implies that it is a promising strategy for tumor-specific therapy for HER2-overexpressing breast cancers.     

Anti-tumor activity of paclitaxel through dual-targeting lipoprotein-mimicking nanocarrier

In the present study, we devised a strategy that paclitaxel (PTX) with lipid and octadecylamine were prepared to lipid nanoparticle (PTX-LNP) with positive charge, folic acid-modified bovine serum albumin (FB)-coated surface of PTX-LNP through electrostatic attraction and generated the lipoprotein-mimicking nanocomplex (FB-PTX-LNP) for dual-targeting therapy. Bovine serum albumin (BSA) was used as the protein model due to its specific targeting to tumor by increased transendothelial gp60-mediated transport and increased intratumoral accumulation as a result of the secreted protein, acidic and rich in cysteine (SPARC)–albumin interaction. The further conjugating folic acid to BSA achieved the dual active targeting. In vitro cytotoxicity tests suggested FB-PTX-LNP and BSA-PTX-LNP exhibited significantly higher cytotoxic activity against MCF-7 and HepG2 cells compared to PTX-LNP. The cellular uptake experiments indicated that FB-coumarin-6-LNP modified with dual-targeting had a faster and greater cellular uptake when compared to BSA-coumarin-6-LNP and coumarin-6-LNP by MCF-7 cells. Thus, both BSA and FA did play roles in in vitro cytotoxicity and cellular uptake. Furthermore, the targeting ability and therapeutic efficacy of FB-PTX-LNP were assessed in vivo. FB-PTX-LNP produced very marked targeting ability and anti-tumor activity in MDA-MB-231 tumor-bearing mice. These results indicate the protein–lipid nanocomplex FB-PTX-LNP is a potential nanocarrier for Paclitaxel dual-targeting to tumor.     

INO-1001, a novel inhibitor of poly(ADP-ribose) polymerase,enhances tumor response to doxorubicin

Summary Poly(ADP-ribose) synthetase inhibitor, INO-1001, is known to sensitize cells to radiation in vitro by inhibiting the repair of DNA damage. Recent evidence has suggested that PARP inhibition may also be a way of selectively targeting p53 deficient cancer cells. The present study tested INO-1001 for its in vivo effect on the chemoresponse of two p53 deficient tumors, human breast cancer MDA-MB-231 and murine mammary carcinoma MCa-K. Doxorubicin was used as the DNA damaging agent and tumor growth delay assay was used as the endpoint. Results showed that INO-1001 was highly effective in enhancing the anti-tumor effects of Doxorubicin for both MDA-MB-231 (EF = 1.88) and MCa-K (EF = 1.64). We conclude that PARP inhibitor INO-1001 has high potential for enhancing the anti-tumor effects of chemotherapy agents such as Doxorubicin against p53 deficient breast cancer.     

Immunoliposomal delivery of doxorubicin can overcome multidrug resistance mechanisms in EGFR-overexpressing tumor cells

Immunoliposomes (ILs) can be constructed to target the epidermal growth factor receptor (EGFR) to provide efficient intracellular drug delivery in tumor cells. We hypothesized that this approach might be able to overcome drug resistance mechanisms, which remain an important obstacle to better outcomes in cancer therapy. ILs were evaluated in vitro and in vivo against EGFR-overexpressing pairs of human cancer cells (HT-29 and MDA-MB-231) that either lack or feature the multidrug resistance (mdr) phenotype. In multidrug-resistant cell lines, ILs loaded with doxorubicin (DOX) produced 19–216-fold greater cytotoxicity than free DOX, whereas in nonresistant cells, immunoliposomal cytotoxicity of DOX was comparable with that of the free drug. In intracellular distribution studies, free DOX was efficiently pumped out of the multidrug-resistant tumor cells, whereas immunoliposomal DOX leads to 3.5–8 times higher accumulation of DOX in the cytoplasm and 3.5–4.9 times in the nuclei compared with the free drug. Finally, in vivo studies in the MDA-MB-231 Vb100 xenograft model confirmed the ability of anti-EGFR ILs-DOX to efficiently target multidrug-resistant cells and showed impressive antitumor effects, clearly superior to all other treatments. In conclusion, ILs provide efficient and targeted drug delivery to EGFR-overexpressing tumor cells and are capable of completely reversing the multidrug-resistant phenotype of human cancer cells.     

Preferential cytotoxicity of the type I ribosome inactivating protein alpha-momorcharin on human nasopharyngeal carcinoma cells under normoxia and hypoxia

All primary nasopharyngeal carcinoma (NPC) tumors contain hypoxic regions which are implicated in decreased local control and increased distant metastases, as well as resistance to chemotherapy in advanced NPC patients. One of the promising therapeutic approaches for NPC is to use drugs that can target hypoxic factors in tumors. In the present investigation, the type I ribosome inactivating protein α-momorcharin (α-MMC), isolated from seeds of the bitter gourd Momordica charantia, reduced cell viability and inhibited clonogenic formation of human NPC CNE2 and HONE1 cells under normoxia and cobalt chloride-induced hypoxia. By comparison, α-MMC exhibited only slight cytotoxicity on human nasopharyngeal epithelial NP69 cells under normoxia. Interestingly, α-MMC suppressed the expression levels of hypoxia-inducible factor 1-alpha (HIF1α) and vascular endothelial growth factor (VEGF) in hypoxic NPC, as well as the growth of human umbilical vein endothelial cells. Further study disclosed that α-MMC targeted endoplasmic reticulum and down-regulated unfolded protein response (UPR) in NPC cells. Moreover, α-MMC induced apoptosis in NPC cells in a dose- and time-dependent manner. It initiated mitochondrial- and death receptor-mediated apoptotic signaling in CNE2 cells, but there was hardly any effect on HONE1 cells. In addition, α-MMC brought about G0/G1 phase cell cycle arrest in CNE2 cells and S phase arrest in HONE1 cells. Collectively, α-MMC preferentially exhibited inhibitory effect on normoxic and hypoxic NPC cells partly by blocking survival signaling (e.g. HIF1α, VEGF and UPR), and triggering apoptotic pathways mediated by mitochondria or death receptor. These observations indicate the potential utility of α-MMC for prophylaxis and therapy of NPC.     

Noninvasive Monitoring of HPMA Copolymer–RGDfK Conjugates by Magnetic Resonance Imaging

Purpose  To evaluate the tumor targeting potential of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–gadolinium(Gd)–RGDfK conjugates by magnetic resonance (MR) T1-mapping. Methods  HPMA copolymers with and without RGDfK were synthesized to incorporate side chains for Gd chelation. The conjugates were characterized by their side-chain contents and r₁ relaxivity. In vitro integrin-binding affinities of polymeric conjugates were assessed via competitive cell binding assays on HUVEC endothelial cells and MDA-MB-231 breast cancer cells. In vivo MR imaging was performed on MDA-MB-231 tumor-bearing SCID mice at different time points using non-targetable and targetable polymers. The specificity of αvβ3 targeting was assessed by using non-paramagnetic targetable polymer to block αvβ3 integrins followed by injection of paramagnetic targetable polymers after 2 h. Results  The polymer conjugates showed relaxivities higher than Gd-DOTA. Endothelial cell binding studies showed that IC₅₀ values for the copolymer with RGDfK binding to αvβ3 integrin-positive HUVEC and MDA-MB-231 cells were similar to that of free peptide. Significantly lower T1 values were observed at the tumor site after 2 h using targetable conjugate (p < 0.012). In vivo blocking study showed significantly higher T1 values (p < 0.045) compared to targetable conjugate. Conclusion  These results demonstrate the potential of this conjugate as an effective targetable MR contrast agent for tumor imaging and therapy monitoring.     

In-vivo anti-tumor activity of a novel poloxamer-based thermosensitive in situ gel for sustained delivery of norcantharidin

In order to develop a novel norcantharidin (NCTD) delivery system with slow drug release and specific targeting characteristics, we have developed a Poloxamer-based NCTD thermosensitive in situ gel. The evaluation of the characteristics of this system using both in vitro and in vivo methods was previously reported. However, its anti-tumor activity in vivo is still not confirmed. Thus, the potential anti-tumor activity and relative mechanism were investigated in a murine H22 hepatoma model. Tumor-bearing mice were treated with different dose of NCTD thermosensitive in situ gel (3.3?mg/kg, 6.6?mg/kg, and 9.9?mg/kg, respectively by intra-tumor injection once every three days, totaling 5 injections per group. Control groups included untreated or NCTD injection (2.2?mg/kg, qd) or blank in situ gel. The expression of vascular endothelial growth factor (VEGF) and CD44 in tumor tissue was examined by immunohistochemistry (IHC) staining. Treatment with middle or high dose of NCTD thermosensitive in situ gel significantly induced tumor regression, inhibited VEGF and CD44 expression and improved survival of tumor-bearing mice. The efficacy of NCTD thermosensitive in situ gel is higher than that of free NCTD injection. Therefore, NCTD thermosensitive in situ gel is a novel NCTD delivery approach for chemotherapeutic treatment of cancer.     

Anti-tumor effect of ultrasound-induced Nordy-loaded microbubbles destruction

Background: Synthesized dl-Nordihydroguaiaretic acid (dl-NGDA or “Nordy”) can inhibit the growth of malignant human tumors, especially the tumor angiogenesis. However, its liposoluble nature limits its in vivo efficacy in the hydrosoluble circulation of human.

Purpose: We tried to use the ultrasonic microbubble as the carrier and the ultrasound-induced destruction for the targeted release of Nordy and evaluate its in vitro and in vivo anti-tumor effect.

Methods: Nordy-loaded lipid microbubbles were prepared by mechanical vibration. Effects of ultrasound-induced Nordy-loaded microbubbles destruction on proliferation of human umbilical vein endothelial cells (HUVECs), tumor derived endothelial cells (Td-ECs), and rabbit transplanted VX2 tumor models were evaluated.

Results: The ultrasound-induced Nordy-loaded microbubbles destruction inhibited the proliferations of HUVECs and Td-ECs in vitro, and inhibited the tumor growth and the microvasculature in vivo. Its efficacy was higher than those of Nordy used only and Nordy with ultrasound exposure.

Conclusion: Ultrasonic microbubbles can be used as the carrier of Nordy and achieve its targeted release with improved anti-tumor efficacy in the condition of ultrasound-induced microbubbles destruction.     

Penetration depth,concentration and efficiency of transdermal α-arbutin delivery after ultrasound treatment with albumin-shelled microbubbles in mice

Recently, the feasibility and effects of using microbubbles (MBs) as an ultrasound (US) contrast agent for enhancing the penetration in transdermal delivery in vivo have been demonstrated, but the mechanism and efficiency are unclear. This study demonstrates the penetration depth, concentration and efficiency of transdermal α-arbutin delivery during 4 weeks after US treatment with MBs in mice. Experimental animals were randomly divided into the following four groups (n?=?5 animals per group): (1) penetrating α-arbutin alone (C), (2) US combined with penetrating α-arbutin, (3) US combined with MBs and penetrating α-arbutin, and (4) US combined with diluted MBs and penetrating α-arbutin (UBD). The penetration depths in agarose phantoms and pigskin were 47 and 84% greater for group UBD, respectively, than for group C. The in vitro skin penetration by 2% α-arbutin after 3?h was 83% greater in group UBD than in group C. The degree of in vivo skin whitening (quantified as the luminosity index) in group UBD significantly increased by 25% after 1 week, 34% after 2 weeks, and then stabilized after 3 weeks at 37% in C57BL/6J mice over a 4-week experimental period. Our results indicate that combined treatment with optimal US and MBs can increase skin permeability so as to enhance α-arbutin delivery to inhibit melanogenesis without damaging the skin in mice.