Enhanced Antitumor Efficacy and Reduced Systemic Toxicity of Sulfatide-Containing Nanoliposomal Doxorubicin in a Xenograft Model of Colorectal Cancer

Sulfatide is a glycosphingolipid known to interact with several extracellular matrix proteins, such as tenascin-C which is overexpressed in many types of cancer including that of the colon. In view of the limited success of chemotherapy in colorectal cancer and high toxicity of doxorubicin (DOX), a sulfatide-containing liposome (SCL) encapsulation approach was taken to overcome these barriers. This study assessed the in vitro cytotoxicity, biodistribution, therapeutic efficacy and systemic toxicity in vivo of sulfatide-containing liposomal doxorubicin (SCL-DOX) using human colonic adenocarcinoma HT-29 xenograft as the experimental model. In vitro, SCL-DOX was shown to be delivered into the nuclei and displayed prolonged retention compared with the free DOX. The use of this nanodrug delivery system to deliver DOX for treatment of tumor-bearing mice produced a much improved therapeutic efficacy in terms of tumor growth suppression and extended survival in contrast to the free drug. Furthermore, treatment of tumor-bearing mice with SCL-DOX resulted in a lower DOX uptake in the principal sites of toxicity of the free drug, namely the heart and skin, as well as reduced myelosuppression and diminished cardiotoxicity. Such natural lipid-guided nanodrug delivery systems may represent a new strategy for the development of effective anticancer chemotherapeutics targeting the tumor microenvironment for both primary tumor and micrometastases.     

跑台干预对多柔比星抗肿瘤效果和心毒性的影响

本研究旨在探讨利用多柔比星进行抗肿瘤治疗前先行跑台干预,对多柔比星抗肿瘤效果及其诱发的心毒性可能造成的影响。本研究采用50只12周龄的C57BL/6小鼠为实验对象,随机分为C组(对照组,n=10)、SNTD组(不运动+无肿瘤+多柔比星组, n=10)、STS组(不运动+肿瘤+安慰剂, n=10)、STD组(不运动+肿瘤+多柔比星组, n=10)、ETD组(运动+肿瘤+多柔比星组, n=10),进行2周的不同方案干预后,在STS组、STD组、ETD组大鼠注射黑色素肿瘤细胞,C组、ETD组注射等量的生理盐水。注射后3周,在SNTD组、STD组、ETD组小鼠的腹腔注射12 mg/kg的多柔比星,C组和STS组注射等量生理盐水。注射结束10 d后,进行心脏形态功能和肿瘤体积检查,利用SPSS进行统计分析。本研究认为多柔比星能够抑制肿瘤流体生长速度,但跑台运动不会对多柔比星抗肿瘤的效果产生影响。注射多柔比星会导致心壁变薄、心输出量、射血量等心脏功能衰退,但跑台运动能够缓解多柔比星的心毒性。     

A Mathematical Model of Breast Cancer Treatment with CMF and Doxorubicin

A mathematical model is presented to investigate the ordering phenomenon observed in the comparison of alternating to sequential regimens of CMF (cyclophosphamid, methotrexate, 5-fluorouracil) and doxorubicin used in breast cancer chemo-therapy. The ordinary differential equation model incorporates cell cycle specificity and resistance to study why doses of the same drugs given in different orders result in different clinical outcomes. The model employs a pulsing condition to simulate treatment and induced resistance, and we investigate treatment outcome by simulating a patient population by varying parameters using uniform distributions. The results of these simulations correspond to those observed in prior clinical studies and suggest that drug resistance might be a key mechanism in the sequential regimen’s superiority.     

miR146a通过Smad4参与多柔比星的心肌细胞毒性作用

目的:探讨miR146a参与多柔比星心肌细胞毒性作用的可能机制。方法:用多柔比星处理大鼠心肌细胞,用CCK-8方法检测细胞活力,用荧光定量PCR检测miR146a的变化,用Western blot检测切割的Caspase 3的蛋白变化。使用常间回文重复序列丛集(clustered regularly interspaced short palindromic repeats,CRISPR)的方法,设计针对miR146a的向导RNA,敲除miR146a的表达。用CCK-8方法和Western blot分别检测敲除细胞的活力和Caspase 3蛋白变化。用软件预测miR146a的靶基因,利用荧光素酶系统进行验证。用Western blot检测多柔比星处理后靶基因的变化,以及用Western blot检测敲除miR146a对靶基因变化的影响。结果:多柔比星处理导致大鼠心肌细胞活力降低,切割的Caspase 3水平升高,同时发现miR146a表达升高(3.6倍)。使用CRISPR可有效敲除miR146a的表达,敲除效果显著高于microRNA decoy的效果(88.6%vs 57.6%)。敲除miR146a的细胞用多柔比星处理,miR146a增加不明显(1.08倍),并且敲除miR146a抑制了多柔比星导致的细胞活力降低和Caspase 3升高。经生物信息学及荧光素酶检测证实在大鼠心肌细胞内Smad family member 4(Smad4)是miR146a的靶基因,用多柔比星处理心肌细胞后,Smad4的表达降低。而敲除miR146a后,Smad4的表达升高,用多柔比星处理敲除miR146a的细胞,Smad4的表达变化不明显。结论:大鼠心肌细胞中,miR146a通过调节Smad4的表达参与了多柔比星对细胞的毒性作用。     

The Clinical Efficacy and Cardiotoxicity of Fixed-Dose Monthly Trastuzumab in HER2-Positive Breast Cancer: A Single Institutional Analysis

Objective

Trastuzumab-containing treatment regimens have been shown to improve survival outcomes in HER2-positive breast cancer (BC). It is much easier to infuse a fixed one-vial dose to every patient on a regular schedule in the general clinical setting. The aims of this study were evaluating the efficacy of a 440 mg fixed-dose of trastuzumab administered on a monthly infusion schedule, and the risk factors for cardiac events.

Patients and methods

We retrospectively reviewed data from 300 HER2-positive BC patients in our institute: 208 were early-stage BC patients undergoing adjuvant trastuzumab treatment, and 92 were metastatic BC patients treated with trastuzumab infusions until disease progression. There were 181 patients receiving regular trastuzumab infusions every 3 weeks (Q3W; 8 mg/kg loading dose followed by 6 mg/kg every 3 weeks), and the other 119 patients were treated monthly with a fixed 440 mg dose (QM; fixed 440 mg every 4 weeks).

Results

The medians of progression-free survival (PFS) and overall survival (OS) in the adjuvant setting were not reached in both treatment groups. In the metastatic setting, there was no significant difference between groups in PFS or OS. The median time to significant cardiovascular (CV) dysfunction was 4.54 months. The incidence of congestive heart failure requiring medication in our cohort was 3.4%.

Conclusion

In our study, we found that fixed-dose monthly trastuzumab was feasible and effective. In addition, the CV risk was not higher with the fixed-dose protocol. This treatment modality could lower the cost and was easier to implement in clinical practice. Larger prospective randomized studies with longer-term follow up are needed to confirm our results.     

Doxorubicin Influences the Expression of Glucosylceramide Synthase in Invasive Ductal Breast Cancer

Introduction

Glucosylceramide synthase (GCS) is one enzyme that provides a major route for ceramide clearance. Recent evidence has indicated an important role for GCS in multidrug resistance (MDR) tumors. Doxorubicin (DOX)can modulate the expression of GCS in leukemia and ovary cell lines. However, few studies have investigated their relationship in breast cancer;

Methods

We collected 84 excision biopsies from patients with invasive ductal breast cancer of whom 33 patients had undergone preoperative chemotherapy. Immunohistochemistry was used to analyze the expression of GCS protein and significantly showed that the expression of GCS was higher in the samples from patients treated with preoperative chemotherapy(p = 0.018). In order to investigate the underlying mechanism, breast cancer cell lines were cultured with different concentrations of DOX, and mRNA and protein levels of GCS were then detected;

Results

DOX significantly upregulated the expression of GCS at both the mRNA and protein level in ERα-positive MCF-7 cells.We then block down the Sp1 site of GCS promoter, which inhibited the DOX-mediated increase in GCS expression; and after Erα was inhibited in MCF-7 cells, the up-regulation of GCS by DOX also been inhibited.

Conclusions

In conclusion, our data demonstrated the novel finding that DOX could modulate the expression of GCS through the Sp1 site of GCS promoter in ERα-positive breast cancer cells.     

Cold Atmospheric Plasma-Activated Media Improve Paclitaxel Efficacy on Breast Cancer Cells in a Combined Treatment Model

Simple SummaryBreast cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality in females, with chemotherapy currently the only adjunctive therapy. However, the disadvantages of chemotherapy are represented by toxicity/side effects, and for many women the benefit is uncertain. Cold atmospheric plasma in in vitro and in vivo experiments proved that it is able to selectively inhibit the growth of cancer cells in various cancer types, with less deleterious side effects. Current guidelines include paclitaxel as a chemotherapy of choice for various types of breast cancers. The aim of the study was to evaluate in vitro, on human breast cancer cell lines, a plasma-activated media as a cytotoxic agent and as an associative agent in a combined treatment for breast cancer with paclitaxel. The collected data indicated that the plasma-activated media amplified the cytotoxic effect of paclitaxel.AbstractThe use of plasma-activated media (PAM), an alternative to direct delivery of cold atmospheric plasma to cancer cells, has recently gained interest in the plasma medicine field. Paclitaxel (PTX) is used as a chemotherapy of choice for various types of breast cancers, which is the leading cause of mortality in females due to cancer. In this study, we evaluated an alternative way to improve anti-cancerous efficiency of PTX by association with PAM, the ultimate achievement being a better outcome in killing tumoral cells at smaller doses of PTX. MCF-7 and MDA-MB-231 cell lines were used, and the outcome was measured by cell viability (MTT assay), the survival rate (clonogenic assay), apoptosis occurrence, and genotoxicity (COMET assay). Treatment consisted of the use of PAM in combination with under IC50 doses of PTX in short- and long-term models. The experimental data showed that PAM had the capacity to improve PTX’s cytotoxicity, as viability of the breast cancer cells dropped, an effect maintained in long-term experiments. A higher frequency of apoptotic, dead cells, and DNA fragmentation was registered in cells treated with the combined treatment as compared with those treated only with PT. Overall, PAM had the capacity to amplify the anti-cancerous effect of PTX.     

TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O₂ was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O₂ induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.     

Improved Efficacy and Reduced Toxicity of Doxorubicin Encapsulated in Sulfatide-Containing Nanoliposome in a Glioma Model

As a glycosphingolipid that can bind to several extracellular matrix proteins, sulfatide has the potential to become an effective targeting agent for tumors overexpressing tenasin-C in their microenvironment. To overcome the dose-limiting toxicity of doxorubicin (DOX), a sulfatide-containing nanoliposome (SCN) encapsulation approach was employed to improve treatment efficacy and reduce side effects of free DOX. This study analysed in vitro characteristics of sulfatide-containing nanoliposomal DOX (SCN-DOX) and assessed its cytotoxicity in vitro, as well as biodistribution, therapeutic efficacy, and systemic toxicity in a human glioblastoma U-118MG xenograft model. SCN-DOX was shown to achieve highest drug to lipid ratio (0.5∶1) and a remarkable in vitro stability. Moreover, DOX encapsulated in SCN was shown to be delivered into the nuclei and displayed prolonged retention over free DOX in U-118MG cells. This simple two-lipid SCN-DOX nanodrug has favourable pharmacokinetic attributes in terms of prolonged circulation time, reduced volume of distribution and enhanced bioavailability in healthy rats. As a result of the improved biodistribution, an enhanced treatment efficacy of SCN-DOX was found in glioma-bearing mice compared to the free drug. Finally, a reduction in the accumulation of DOX in the drug''s principal toxicity organs achieved by SCN-DOX led to the diminished systemic toxicity as evident from the plasma biochemical analyses. Thus, SCN has the potential to be an effective and safer nano-carrier for targeted delivery of therapeutic agents to tumors with elevated expression of tenascin-C in their microenvironment.     

MicroRNA-155 Deficiency Attenuates Liver Steatosis and Fibrosis without Reducing Inflammation in a Mouse Model of Steatohepatitis

Background & Aim

MicroRNAs (miRs) regulate hepatic steatosis, inflammation and fibrosis. Fibrosis is the consequence of chronic tissue damage and inflammation. We hypothesized that deficiency of miR-155, a master regulator of inflammation, attenuates steatohepatitis and fibrosis.

Methods

Wild type (WT) and miR-155-deficient (KO) mice were fed methionine-choline-deficient (MCD) or -supplemented (MCS) control diet for 5 weeks. Liver injury, inflammation, steatosis and fibrosis were assessed.

Results

MCD diet resulted in steatohepatitis and increased miR-155 expression in total liver, hepatocytes and Kupffer cells. Steatosis and expression of genes involved in fatty acid metabolism were attenuated in miR-155 KO mice after MCD feeding. In contrast, miR-155 deficiency failed to attenuate inflammatory cell infiltration, nuclear factor κ beta (NF-κB) activation and enhanced the expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα) and monocyte chemoattractant protein-1 (MCP1) in MCD diet-fed mice. We found a significant attenuation of apoptosis (cleaved caspase-3) and reduction in collagen and α smooth muscle actin (αSMA) levels in miR-155 KO mice compared to WTs on MCD diet. In addition, we found attenuation of platelet derived growth factor (PDGF), a pro-fibrotic cytokine; SMAD family member 3 (Smad3), a protein involved in transforming growth factor-β (TGFβ) signal transduction and vimentin, a mesenchymal marker and indirect indicator of epithelial-to-mesenchymal transition (EMT) in miR-155 KO mice. Nuclear binding of CCAAT enhancer binding protein β (C/EBPβ) a miR-155 target involved in EMT was significantly increased in miR-155 KO compared to WT mice.

Conclusions

Our novel data demonstrate that miR-155 deficiency can reduce steatosis and fibrosis without decreasing inflammation in steatohepatitis.     

Genetic Profiling of Breast Cancer with and Without Preexisting Metabolic Disease

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women. Various mechanisms are involved in the initiation and progression of breast cancer. Metabolic dysregulation has been associated with increasing breast cancer incidence and mortality. However, little is known about how metabolic disease regulates the development and progression of breast cancer at the molecular level. Here, using a hybridization capture-based panel including 124 cancer-associated genes, we performed targeted next-generation sequencing of tumor tissues and matched blood samples from 20 postmenopausal patients with primary breast cancer, in which 6 cases suffered from preexisting metabolic disorders including hypertension, type 2 diabetes, and coronary heart disease. We took only the protein-altering variants and identified 170 somatic mutations of 59 genes. Among these, 40 mutated genes were found in the metabolic disease group, and 33 mutated genes were found in the non–metabolic disease group. Importantly, nonsynonymous mutations of 26 genes (MSH3, BRAF, MLH3, MTOR, DDR2, ALK, etc.) were uniquely present in the metabolic disease group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to investigate biological functions and key pathways of somatic mutations. TP53, PIK3CA, and PTEN were the top three commonly mutated genes at a higher frequency compared with the Cancer Genome Atlas (TCGA) data, and several novel but infrequent mutations in other genes were also found. Although further studies are required to validate these variants, our results are the first to suggest a specific molecular profile of breast cancer with preexisting metabolic disease.     

Evodiamine Synergizes with Doxorubicin in the Treatment of Chemoresistant Human Breast Cancer without Inhibiting P-Glycoprotein

Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO), a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX)-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose) polymerase (PARP), caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs). Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp). Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.     

Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H₂O₂-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Effect of Transcatheter Intraarterial Therapies on the Distribution of Doxorubicin in Liver Cancer in a Rabbit Model

Background and Aims

Transcatheter intraarterial techniques can effectively deliver chemotherapeutic agents to tumor and improve the efficacy of chemotherapy. The present study is designed to evaluate the effect of transcatheter intraarterial techniques on the distribution of doxorubicin in relation to blood vessels in liver cancer.

Methods

VX2 tumors were implanted in the livers of 32 rabbits. The animals were divided into 4 groups of 8 animals each. Group 1 (doxo iv) animals received doxorubicin intravenous injection; group 2 (doxo ia) received doxorubicin hepatic intraarterial infusion; group 3 (doxo ia + E) received doxorubicin hepatic intraarterial infusion followed by embolization; group 4 (doxo + L ia + E) received hepatic intraarterial infusion of doxorubicin mixed with Lipiodol followed by embolization. Ten minutes or 4 hours after treatment, the animals were sacrificed and tumors were sampled. Immunofluorescence techniques were used to evaluate the distribution of doxorubicin in relation to blood vessels.

Results

Doxorubicin fluorescence was distributed around tumor blood vessels and decreased with distance from the blood vessels. Tumor cells in avascular and adjacent regions were not exposed to detectable concentrations of doxorubicin. Tumors in the group 2, 3 and 4 had a significant increase in doxorubicin penetration compared with the group 1 tumors (P<0.05). Among the three groups of transcatheter therapies, doxorubicin penetration distance in group 3 was significantly larger than that in group 2 and 4 (P<0.05), and no significant difference was found between group 2 and 4 tumors (P>0.05) at 10 minutes. In contrast, at 4 hours and in total, both group 3 and 4 tumors had significant increases in drug penetration compared with group 2 (P<0.05), and no significant difference was noted between group 3 and 4 tumors (P>0.05).

Conclusion

Transcatheter intraarterial therapies improve doxorubicin penetration in liver cancer; nevertheless their effect on drug distribution is somewhat limited.     

Enhanced Delivery and Antitumor Effect of Doxorubicin Encapsulated in Long-Circulating Liposomes

Abstract

Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) or ganglioside GM1 (GM1) have been tested for their utility as enhanced delivery system of doxorubicin (DXR) in vivo. DXR was entrapped into liposomes by pH gradient method.

The long-circulating LUV (200 nm in size) composed of DSPC/CH (1:1, m/m) and either 6 mol% of DSPE-PEG1000 or GM1 entrapped DXR with >95% in trapping efficiency. DXR-long-circulating LUVs were administered to leukemic (LI210) mice via the tail vein at a dose of 5mg DXR/kg. The high blood concentration was kept for long time, and significantly increased survival time was observed as compared with free DXR and DXR-LUV. The data indicated that DXR was slowly released from long-circulating LUV during that stayed in bloodstream for long time. Administration of DXR-long-circulating SUV (100 nm) to the colon 26 bearing mice produced the increased DXR level in tumor compared with bear SUV or free drug did, respectively, and resulted in effective tumor growth retardation and increased survival time. DXR was delivered to tumor by accumulation of SUVs themselves.

Long-circulating thermosensitive liposomes (TSL) were prepared from DPPC /DSPC (9:1, m/m) and 3-6 mol% of PEG1000 or GM1. DXR was entrapped with >95% in trapping efficiency. Accumulation of DXR into tumor tissue by local hyperthermia after injection of DXR-long-circulating TSL to colon 26 bearing mice was significantly higher man that of DXR-bare TSL or free DXR, and resulted in effective tumor growth retardation and increased survival time. It was suggested that the entrapped DXR was efficiently released from long-circulating TSL by hyperthermia at the tumor site and entered the tumor tissue by simple diffusion.     

Nanomicellar Formulation of Clotrimazole Improves Its Antitumor Action toward Human Breast Cancer Cells

Background

Although demonstrated as a selective anticancer drug, the clinical use of clotrimazole (CTZ) is limited due to its low solubility in hydrophilic fluids. Thus, we prepared a water-soluble nanomicellar formulation of CTZ (_nCTZ) and tested on the human breast cancer cell line MCF-7 biology.

Methodology/Principal Findings

CTZ was nanoencapsulated in tween 80 micelles, which generated nanomicelles of, approximately, 17 nm of diameter. MCF-7 cells were treated with _nCTZ and unencapsulated DMSO-solubilized drug (_sCTZ) was used for comparison. After treatment, the cells were evaluated in terms of metabolism, proliferation, survival and structure. We found that _nCTZ was more efficient than _sCTZ at inhibiting glycolytic and other cytosolic and mitochondrial enzymes. Moreover, this increased activity was also observed for lactate production, intracellular ATP content, ROS production and antioxidant potential. As a consequence, _nCTZ-treated MCF-7 cells displayed alterations to the plasma membrane, mitochondria and the nucleus. Finally, _nCTZ induced both apoptosis and necrosis in MCF-7 cells.

Conclusions/Significance

MCF-7 cells are more sensible to _nCTZ than to _sCTZ. This was especially evident on regard to antioxidant potential, which is an important cell defense against drugs that affect cell metabolism. Moreover, this water-soluble formulation of CTZ strengths its potential use as an anticancer medicine.