Gambogic Acid Sensitizes Ovarian Cancer Cells to Doxorubicin Through ROS-Mediated Apoptosis

Ovarian cancer is one human malignancy which has response portly to doxorubicin. The anti-cancer activity of gambogic acid has been tested in in vitro and in vivo studies. In this study, we showed that gambogic acid, a natural compound, could potentiate the anticancer activity of doxorubicin in ovarian cancer through ROS-mediated apoptosis. Platinum-resistant human ovarian cancer cell line (SKOV-3) was treated with gambogic acid, doxorubicin, or the combination of both to investigate cell proliferation and apoptosis. We found that the combination of gambogic acid and doxorubicin causes synergistic loss of cell viability in SKOV-3 cells and this synergistic effect correlated with increased cellular ROS accumulation. Moreover, in vivo results showed that gambogic acid and doxorubicin combination resulted in a synergistic suppressing effect on tumor growth in ovarian cancer mice model. Taken together, the results suggested that doxorubicin in combination with gambogic acid might provide a promising therapeutic strategy to enhance chemosensitivity of ovarian cancer to doxorubicin.     

Interaction of Doxorubicin and Dipalmitoylphosphatidylcholine Liposomes

The interaction between doxorubicin (DOX), an anthracycline antibiotic frequently used in chemotherapy, and zwitterionic dipalmitoylphosphatidylcholine (DPPC) was investigated using Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and rheological measurements. FTIR results showed that DOX shifted the wavenumber of the PO₂ ⁻ band for pure DPPC to a higher wavenumber. This may have been because of the strong interactions between the NH₃ ⁺ group in DOX and the phosphate (PO₂ ⁻) group in the polar head of DPPC. The main transition temperature of DPPC liposomes was slightly shifted to a lower temperature for DPPC liposome-encapsulated DOX. This suggested that DOX had a significant effect on the acyl chains in the DPPC bilayers, and that its presence decreased the transition cooperativity of lipid acyl chains. There was also the appearance of an additional transition peak at nearly 136°C for the DPPC/DOX sample. These interactions between DOX and DPPC phospholipid would cause a decrease in the DPPC liposomes plastic viscosity and increase membrane fluidity. A better understanding of the interactions between DOX and lipid bilayers could help in the design and development of improved liposomal drug delivery systems.     

Doxorubicin Encapsulated in Long-Circulating Thermosensitive Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (TSL, 180-200 nm in mean diameter), prepared from dipalmitoyl phosphatidyl choline (DPPC)/distearoyl phosphatidyl choline (DSPC) (9:1, m/m) and either 3 mol% of amphipathic polyethylene glycol (PEG) with 1000 in average molecular weight or 6 mol% of ganglioside GMI (GMI), with 95-98% entrapping efficiency by the pH gradient method. 57% or 45% of the entrapped DXR was released from PEG/DPPC/DSPC or G_M1/DPPC/DSPC liposomes, respectively, by incubation with 20% serum at 42°C for 5 min. Inclusion of PEG or G_M1 endowed TSL with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system (RES) uptake over 6 hours after injection. Concomitantly, high DXR level in blood was kept for long time.

Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR-long-circulating TSL was 2 times or 7 times higher than that after treatment with DXR-TSL liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the systemic treatment with DXR-long-circulating TSL and hyperthermia resulted in effective tumor growth retardation and increased survival time. These results indicate that the combination of long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

肿瘤干细胞与卵巢癌研究进展

近年来,肿瘤干细胞学说作为肿瘤发生发展的重要原因获得越来越多的认可。肿瘤干细胞是指肿瘤中存在的含量极少、具有无限增殖潜能的干细胞样肿瘤细胞,它们能自我更新、分化、迁徙,是导致肿瘤发生、发展、转移和耐药的重要原因。卵巢癌也可能是卵巢癌干细胞所致的疾病。卵巢癌干细胞的分离鉴定正处于起始阶段,针对卵巢癌干细胞的靶向治疗可能在卵巢癌治疗中具有重要作用,为临床彻底治愈卵巢癌带来希望。     

RBMS2 Chemosensitizes Breast Cancer Cells to Doxorubicin by Regulating BMF Expression

Chemoresistance is closely related to the therapeutic effect and prognosis in breast cancer patients. Increasing evidences demonstrated that RNA binding proteins (RBPs) have notable roles in regulating cancer cell proliferation, metastasis and chemotherapeutic sensitivity. RNA binding motif single stranded interacting protein 2 (RBMS2), an RBP, has been considered to be a tumor suppressor in several cancers. However, its role of doxorubicin sensitivity in breast cancer patients has not yet been fully revealed. Here, we performed doxorubicin cytotoxicity assay, flow cytometry and mouse xenograft model to examine the influence of RBMS2 on doxorubicin sensitization in vitro and in vivo. RIP assay and dual-luciferase reporter assay were performed to explore the relationship between RBMS2 and BMF. Our data demonstrated that upregulation of RBMS2 in breast cancer cells could enhance sensitivity to doxorubicin and promote apoptosis in the presence of doxorubicin, while inhibition of RBMS2 showed an opposite trend. Moreover, this chemosensitizing effect of RBMS2 could be reversed by the inhibition of Bcl-2 modifying factor (BMF). RBMS2 positively regulated BMF expression and increased BMF-induced expression of (cleaved) caspase 3, (cleaved) caspase 9 and poly (ADP-Ribose) polymerase (PARP). These results uncovered a novel mechanism for RBMS2 in the sensibilization of doxorubicin, suggesting that RBMS2 may act as a potential therapeutic target for drug-resistant breast cancer.     

2-Amino-Pyrrole-Carboxylate Attenuates Homology-Mediated DNA Repair and Sensitizes Cancer Cells to Doxorubicin

Biochemistry (Moscow) - Despite a high efficacy of chemotherapy in cancer treatment, acquired resistance of tumors to certain chemotherapeutic agents and frequent side effects remain the major...     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

Preclinical Studies with Doxorubicin Encapsulated in Polyethyleneglycol-Coated Liposomes

Abstract

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These preclinical observations, suggesting that encapsulation of DOX in PEG-coated liposomes may lead to a significant improvement of the therapeutic index of DOX, have led to the initiation of clinical trials in cancer patients.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Modulation of Multidrug Resistance in Cancer Cells by Liposome Encapsulated Doxorubicin

Abstract

A major problem in the chemotherapy of solid tumors and hematologic malignancies is the intrinsic as well as acquired cross resistance to multiple chemotherapeutic agents. Recently, this type of multidrug resistance has been related to a gene, MDR1, and its gene product, p-glycoprotein, which functions as the efflux pump, prevents accumulation of drugs and alters their cytotoxicity. Many drug-resistant human tumors express the MDR1 gene and MDR1 RNA levels are elevated in many cancers that have not responded to chemotherapy. The same persistent observation has been made in recurrent tumors who have responded initially to chemotherapy.

Doxorubicin is one of the most important anticancer agent having significant single agent activity in a variety of cancer types and is now the cornerstone of some widely used combination regimens. Despite the clinical effectiveness of the drug, doxorubicin resistance that arises in malignant cells following repeated courses of treatment is the major problem in the clinical management of neoplastic diseases. Recently, extensive studies have demonstrated that liposome encapsulated doxorubicin effectively modulates the multidrug resistance phenotype in cancer cells by altering the function of p-glycoprotein. This modulation of MDR phenotype by liposomes has been demonstrated in vitro in human breast cancer cells, ovarian cancer cells, human promyelocytic leukemia cells and in human colon cancer cells and in vivo in transgenic mice transfected with a functional MDR1 gene. It appears liposomes can play an effective role as a new modality of treatment for human cancers which have become refractory to chemotherapy. An exciting area of research which soon will emerge will exploit the different binding sites on p-glycoprotein by using combination of liposomes with other pharmacological modulators of MDR to impart maximal overcoming of multidrug resistance in cancer patients.     

Visualization of Ovarian Cancer Cells with Peptide VEGEGEEGEEY

The binding of peptide VEGEGEEGEEY to the surface of ovarian cancer cells was studied by confocal microscopy. It has been demonstrated that the peptide competes with hyaluronan for the binding to RHAMM (receptor for hyaluronan-mediated motility). It was found that peptide VEGEGEEGEEY specifically bound to RHAMM on ovarian cancer cell surface, and this interaction was blocked by anti-RHAMM antibodies. The peptide did not bind to RHAMM-knockout fibroblasts RHAMM^(–/–) but interacted with transfected fibroblasts RHAMM^(+/+), which further confirms the binding of the peptide to RHAMM. Evaluation of the peptide binding selectivity showed that the peptide reacts with cancer cells and does not bind to the surface of fibroblasts and normal cells. Thus, the specificity of binding of peptide FITC-VEGEGEEGEEY to RHAMM suggests a possibility of its application as a molecular probe for imaging and diagnosis of ovarian cancer at early stages.     

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.     

Withaferin A Synergizes the Therapeutic Effect of Doxorubicin through ROS-Mediated Autophagy in Ovarian Cancer

Application of doxorubicin (Dox) for the treatment of cancer is restricted due to its severe side effects. We used combination strategy by combining doxorubicin (Dox) with withaferin A (WFA) to minimize the ill effects of Dox. Treatment of various epithelial ovarian cancer cell lines (A2780, A2780/CP70 and CaOV3) with combination of WFA and Dox (WFA/DOX) showed a time- and dose-dependent synergistic effect on inhibition of cell proliferation and induction of cell death, thus reducing the dosage requirement of Dox. Combination treatment resulted in a significant enhancement of ROS production resulting in immense DNA damage, induction of autophagy analyzed by transmission electron microscope and increase in expression of autophagy marker LC3B, and culminated in cell death analyzed by cleaved caspase 3. We validated combination therapy on tumor growth using an in vitro 3Dimension (3D) tumor model and the more classic in vivo xenograft model of ovarian cancer. Both tumor models showed a 70 to 80% reduction in tumor growth compared to control or animals treated with WFA or Dox alone. Immunohistochemical analysis of the tumor tissues from animals treated with WFA/Dox combination showed a significant reduction in cell proliferation and formation of microvessels accompanied by increased in LC3B level, cleaved caspase 3, and DNA damage. Taken together, our data suggest that combining WFA with Dox decreases the dosage requirement of Dox, therefore, minimizing/eliminating the severe side effects associated with high doses of DOX, suggesting the application of this combination strategy for the treatment of ovarian and other cancers with no or minimum side effects.     

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.     

LncRNA AC006449.2抑制卵巢癌细胞增殖

长链非编码RNAs(long non-coding RNAs, lncRNAs)是一类无蛋白质编码功能的RNAs。多项研究表明,lncRNAs在肿瘤的发生发展中发挥重要的作用。GEPIA数据库结合qRT-PCR结果提示,lncRNA AC006449.2在卵巢癌组织中的表达量显著低于正常组织,且其在卵巢癌细胞中的水平也低于正常卵巢上皮细胞。进一步分析发现,lncRNA AC006449.2的表达量与卵巢癌患者较差的预后负相关,与瘤体大小和远端转移密切相关,而与卵巢癌患者年龄、淋巴结转移及TNM分期无关。细胞功能研究发现,上调AC006449.2,卵巢癌细胞的增殖和平板克隆能力降低;而下调AC006449.2之后,该细胞的增殖和平板克隆能力显著增加。深入的机制研究发现,AC006449.2可上调细胞周期相关蛋白p21和p27的表达量,上调促凋亡蛋白Bax的表达,而下调抗凋亡蛋白Bcl2的表达量,最终抑制卵巢癌细胞的增殖。上述研究结果推测,lncRNA AC006449.2在卵巢癌细胞中可能发挥抑癌因子的作用。     

Methylseleninic Acid Sensitizes Notch3-Activated OVCA429 Ovarian Cancer Cells to Carboplatin

Ovarian cancer, the deadliest of gynecologic cancers, is usually not diagnosed until advanced stages. Although carboplatin has been popular for treating ovarian cancer for decades, patients eventually develop resistance to this platinum-containing drug. Expression of neurogenic locus notch homolog 3 (Notch3) is associated with chemoresistance and poor overall survival in ovarian cancer patients. Overexpression of NICD3 (the constitutively active form of Notch3) in OVCA429 ovarian cancer cells (OVCA429/NICD3) renders them resistance to carboplatin treatment compared to OVCA429/pCEG cells expressing an empty vector. We have previously shown that methylseleninic acid (MSeA) induces oxidative stress and activates ataxia-telangiectasia mutated and DNA-dependent protein kinase in cancer cells. Here we tested the hypothesis that MSeA and carboplatin exerted a synthetic lethal effect on OVCA429/NICD3 cells. Co-treatment with MSeA synergistically sensitized OVCA429/NICD3 but not OVCA429/pCEG cells to the killing by carboplatin. This synergism was associated with a cell cycle exit at the G2/M phase and the induction of NICD3 target gene HES1. Treatment of N-acetyl cysteine or inhibitors of the above two kinases did not directly impact on the synergism in OVCA429/NICD3 cells. Taken together, these results suggest that the efficacy of carboplatin in the treatment of high grade ovarian carcinoma can be enhanced by a combinational therapy with MSeA.     

Liposomes to Target Peripheral Neurons and Schwann Cells

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.