Increase of therapeutic activity of doxorubicin by long circulating liposomes in combination with curcumin

In this study, doxorubicin (DOX)-loaded long circulating liposomes combined with curcumin (CUR) (DOX-CUR-LCLs) were successfully prepared as a novel formulation for cancer treatment. The particle size and distribution, zeta potential, drug loading capacity, and entrapment efficiency (EE) of the preparation were characterized. The in vitro anti-tumor activities of DOX-CUR-LCLs and DOX-LCLs against A549 cells were then evaluated and compared with that of free DOX. Cytotoxicity evaluation showed that DOX-CUR-LCLs had a significantly higher antitumor activity than other DOX preparations. These results suggest that novel DOX-CUR-LCLs, combination of DOX and CUR administered in long-circulating liposomes, could improve antitumor activity.     

The effective combination therapy against human osteosarcoma: doxorubicin plus curcumin co-encapsulated lipid-coated polymeric nanoparticulate drug delivery system

Objective: To overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure incurred from multidrug resistant (MDR) in osteosarcoma (OS), biodegradable lipid-coated polymeric nanoparticles (LPNs) were explored for the loading of doxorubicin (DOX) and curcumin (CUR).

Methods: DOX plus CUR co-encapsulated LPNs (DOX?+?CUR LPNs) of mixed lipid monolayer shell and biodegradable polymer core were prepared. The cytotoxicity effect of DOX?+?CUR LPNs, single drug loaded LPNs, and free drug solutions were evaluated on human OS cell line KHOS cells and mice KHOS cells xenograft in vivo.

Results: DOX?+?CUR LPNs displayed a curative effect on OS cell lines than the free drug counterparts. Also, best anti-OS effects were observed on the animal model compared with other groups tested.

Conclusion: This promising dual drugs co-encapsulated lipid-coated polymeric nanoparticulate drug delivery system enhanced the cell delivery and activity of drugs against human OS cancer cell lines and in cancer bearing mice. This research may offer new options for the treatment of OS.     

Star polyester-based folate acid-targeting nanoparticles for doxorubicin and curcumin co-delivery to combat multidrug-resistant breast cancer

Chemotherapeutic treatments are indispensable in the treatment of breast cancer. However, the emergence of multidrug-resistance, strong cell toxicity, and poor targeting selection has inhibited their clinical application. In this study, two synergistic drugs, doxorubicin (DOX) and curcumin (CUR), were co-administered to overcome multidrug resistance (MDR). Based on the characteristics of the tumor microenvironment, we developed folic acid-modified nanoparticles ((DOX + CUR)-FA-NPs) based on a star-shaped polyester (FA-TRI-CL) to enhance the tumor targeting selectivity and drug loading (DL) capacity. The (DOX + CUR)-FA-NPs displayed a characteristic spheroid morphology with an ideal diameter (186.52 nm), polydispersity index (0.024), zeta potential (–18.87 mV), and good entrapment efficiency (97.64%/78.13%, DOX/CUR) and DL (20.27%/11.29%, DOX/CUR) values. In vitro pharmacokinetic and pharmacodynamic experiments demonstrated that the (DOX + CUR)-FA-NPs were gradually released, and they displayed the highest cell apoptosis and cellular uptake in MCF-7/ADR cells. Additionally, in vivo results illustrated that (DOX + CUR)-FA-NPs not only displayed significant tumor targeting and anticancer efficacy, but also induced less pathological damage to the normal tissue. In summary, co-administered DOX and CUR appeared to reverse MDR, and this targeted combinational nanoscale delivery system could thus be a promising carrier for tumor therapies in the future.     

Selection of P-Glycoprotein Inhibitor and Formulation of Combinational Nanoformulation Containing Selected Agent Curcumin and DOX for Reversal of Resistance in K562 Cells

Purpose

To select P-glycoprotein (P-gp) inhibitor from natural source for reversal of DOX resistance in K562 cells and to develop selected one in to nanoformulation in combination with DOX.

Methods

DOX resistant K562 (K562R) cells were developed and reversal of resistance by P-gp inhibitor was validated by co-treatment with verapamil. The p-gp inhibitors were evaluated for their potential to inhibit P-gp (calcein assay) and to reverse drug resistance (XTT cell viability assay). The selected agent, curcumin was formulated in to liposome along with DOX and characterized for size, zeta potential, encapsulation efficiency and release rate. Uptake, P-gp inhibition and reversal of acquired drug resistance in K562R cells were performed.

Results

P-gp inhibitors such as biochanin-A and curcumin were marked suitable for combination with DOX. However, only curcumin could increase the sensitivity of DOX at all dosing levels, therefore used for further studies. Liposomes loaded with curcumin were formulated and characterized where a prolonged release was observed. The uptake of liposomal curcumin was comparable to nanodispersed curcumin but had lower cytotoxicity. DOX and curcumin coloaded liposomes successfully reversed DOX resistance in K562 cells. Conclusion: The coloaded liposomes increased the safety of curcumin with improved efficacy thus can be employed for reversal of acquired DOX resistance.

     

Coformulation of doxorubicin and curcumin in poly(D,L-lactide-co-glycolide) nanoparticles suppresses the development of multidrug resistance in K562 cells

Doxorubicin (DOX) is a broad-spectrum anthracycline antibiotic used to treat a variety of cancers including leukemia. Chronic myeloid leukemia (CML) blasts like K562 cells are resistant to apoptosis induced by DOX due to several reasons, the primary being the sequestration of drug into cytoplasmic vesicles and induction of multidrug resistance (MDR) gene expression with DOX treatment resulting in intracellular resistance to this drug. Moreover, expression of antiapoptotic protein BCL-2 and the hybrid gene bcr/abl in K562 cells contributes resistance to DOX. Studies have shown that curcumin (CUR) has a pleiotropic therapeutic effect in cancer treatment, as it is an inhibitor of nuclear factor kappa B (NFκB) as well as a potent downregulator of MDR transporters. In this study, we investigated the potential benefit of using DOX and CUR in a single nanoparticle (NP) formulation to inhibit the development of drug resistance for the enhancement of antiproliferative activity of DOX in K562 cells. Results illustrate that the dual (DOX+CUR) drug loaded NPs were effectively delivered into K562 cells. CUR not only facilitates the retention of DOX in nucleus for a longer period of time but also inhibits the gradual expression of MDR1 and BCL-2 at the mRNA level in K562 cells. Moreover, Western blot results confirm that in combination both of the drugs were capable of inducing apoptosis even if in a lower concentration compared to either single drug in both solution or in formulation. Combinational therapy by using DOX and CUR, especially when administered in the NP formulation, has enhanced the cytotoxicity in K562 cells by promoting the apoptotic response. Overall, this combinational strategy has significant promise in the clinical management of intractable diseases, especially leukemia.     

Anti-cancer activity of anti-GLUT1 antibody-targeted polymeric micelles co-loaded with curcumin and doxorubicin

Abstract

Background: Treatment of late stage cancers has proven to be a very difficult task. Targeted therapy and combinatory drug administration may be the solution.

Purpose: The study was performed to evaluate the therapeutic efficacy of PEG-PE micelles, co-loaded with curcumin (CUR) and doxorubicin (DOX), and targeted with anti-GLUT1 antibody (GLUT1) against HCT-116 human colorectal adenocarcinoma cells both in vitro and in vivo.

Methods: HCT-116 cells were treated with non-targeted and GLUT1-targeted CUR and DOX micelles as a single agent or in combination. Cells were inoculated in female nude mice. Established tumors were treated with the micellar formulations at a dose of 4?mg/kg CUR and 0.4?mg/kg DOX every 2?d for a total of 7 injections.

Results: CUR?+?DOX-loaded micelles decorated with GLUT1 had a robust killing effect even at low doses of DOX in vitro. At the doses chosen, non-targeted CUR and CUR?+?DOX micelles did not exhibit any significant tumor inhibition versus control. However, GLUT1-CUR and GLUT1-CUR?+?DOX micelles showed a significant tumor inhibition effect with an improvement in survival.

Conclusion: We showed a dramatic improvement in efficacy between the non-targeted and GLUT1-targeted formulations both in vitro and in vivo. Hence, we confirmed that GLUT1-CUR?+?DOX micelles are effective and deserve further investigation.     

Folate-modified poly(2-ethyl-2-oxazoline) as hydrophilic corona in polymeric micelles for enhanced intracellular doxorubicin delivery

The transmembrane transport of drug loaded micelles to intracellular compartment is quite crucial for efficient drug delivery. In the current study, we investigated the cellular internalization and anticancer activity of doxorubicin loaded micelles with folate modified stealthy PEOz corona. Folate-decorated micelles incorporating doxorubicin were characterized for particle size, degree of folate decoration, drug loading content and encapsulation efficiency, morphology, and surface charge. The targeting capability and cell viability were assessed using HeLa, KB, A549 and MCF-7/ADR cell lines. In vitro study clearly illustrated the folate receptor (FR) mediated targeting of FA modified micelles to FR-positive human HeLa, KB and MCF-7/ADR cells, while specific delivery to FR-negative A549 cells was not apparently increased at the same experimental conditions. Cytotoxicity assay showed 60% and 58% decrease in IC₅₀ values for HeLa and KB cells, while only a slight decrease for A549 cells, following treatment with folate modified formulations. The enhanced intracellular delivery of FA modified micelles in MCF-7/ADR cells was also observed. In vivo antitumor tests revealed DOX entrapped FA-PEOz-PCL micelles effectively inhibited the tumor growth and reduced the toxicity to mice compared with free DOX. The current study showed that the targeted nano-vector improved cytotoxicity of DOX and suggested that this novel PEOz endowed stealthy micelle system held great promise in tumor targeted therapy.     

Single-walled carbon nanotube-loaded doxorubicin and Gd-DTPA for targeted drug delivery and magnetic resonance imaging

An aspargine-glycine-arginine (NGR) peptide modified single-walled carbon nanotubes (SWCNTs) system, developed by a simple non-covalent approach, could be loaded with the anticancer drug doxorubicin (DOX) and magnetic resonance imaging (MRI) contrast agent gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). This DOX- and Gd-DTPA-loaded NGR functionalized SWCNTs (DOX/NGR-SWCNTs/Gd-DPTA) retained both cytotoxicity of DOX and MRI contrast effect of Gd-DPTA. This drug delivery system showed excellent stability in physiological solutions. This DOX/NGR-SWCNTs/Gd-DPTA system could accumulate in tumors and enter into tumor cells, which facilitated combination chemotherapy with diagnosis of tumor in one system. An excellent in vitro anti-tumor effect was shown in MCF-7 cells treated by DOX/NGR-SWCNTs/Gd-DPTA, compared with DOX solution, DOX/SWCNTs and DOX/SWCNTs/Gd-DPTA. In vivo data of DOX/NGR-SWCNTs/Gd-DPTA group in tumor-bearing mice further confirmed that this system performed much higher tumor targeting capacity and anti-tumor efficacy than other control groups.     

Temperature and pH-responsive PNIPAM@PAA Nanospheres with a Core-Shell Structure for Controlled Release of Doxorubicin in Breast Cancer Treatment

Stimuli-responsive polymers have been of great interest in the fabrication of advanced drug delivery systems. In this study, a facile approach was developed to synthesize a dually temperature/pH-responsive drug delivery system with a core-shell structure to control the release of doxorubicin (DOX) at the target site. For this purpose, poly(acrylic acid) (PAA) nanospheres were first synthesized using the precipitation polymerization technique and were used as pH-responsive polymeric cores. Then, poly(N-isopropylacrylamide) (PNIPAM) with thermo-responsivity properties was coated on the outer surface of PAA cores via seed emulsion polymerization technique to render monodisperse PNIPAM-coated PAA (PNIPAM@PAA) nanospheres. The optimized PNIPAM@PAA nanospheres with an average particle size of 116.8 nm (PDI= 0.243), had a high negative surface charge (zeta potential= -47.6 mV). Then, DOX was loaded on PNIPAM@PAA nanospheres and the entrapment efficiency (EE) and drug loading (DL) capacity were measured to be 92.7% and 18.5%, respectively. The drug-loaded nanospheres exhibited a low leakage at neutral pH and physiological temperature, but drug release significantly enhanced at acidic pH (pH= 5.5), indicating the tumor-environment responsive drug release behavior of the prepared nanospheres. Also, kinetics studies showed that, the sustained release of DOX from PNIPAM@PAA nanospheres was consistent with the Fickian diffusion mechanism. Moreover, the anticancer efficacy of DOX-loaded nanospheres was evaluated in vitro against MCF-7 breast cancer cells. The obtained results revealed that, the incorporation of DOX into PNIPAM@PAA nanospheres increases its cytotoxicity against cancer cells compared to the free DOX. Our results suggest that, PNIPAM@PAA nanospheres can be considered as a promising vector to release anticancer drugs with dual-stimuli responsivity to pH and temperature.     

Reversal activity of nanostructured lipid carriers loading cytotoxic drug in multi-drug resistant cancer cells

To overcome multi-drug resistance (MDR) of cancer cells, paclitaxel (PTX) and doxorubicin (DOX)-loaded nanostructured lipid carriers (NLC) were prepared by solvent diffusion method using monostearin as solid lipid and oleic acid as liquid lipid matrix. The cytotoxicities and reversal activity of drug-loaded NLC were tested against human breast cancer (MCF-7) cells, human ovarian cancer (SKOV3) cells and their multi-drug resistant (MCF-7/ADR and SKOV3-TR30) cells. The chemical conjugant of folic acid and stearic acid (FA-SA) was further synthesized to prepare folated NLC. Comparing with taxol and doxorubicin solution, the NLC loading PTX exhibited high cytotoxicities in MCF-7 and MCF-7/ADR cells, while the NLC loading DOX only indicated high cytotoxicity in MCF-7/ADR cells. The reversal powers of the NLC loading PTX and DOX were 34.3 and 6.4 folds, respectively. The NLC loading PTX and DOX showed the same trends of enhanced cytotoxicity against SKOV3 and SKOV3-TR30 cells. The reversal powers were 31.3 and 2.2 folds for the NLC loading PTX and DOX, respectively. The modification of NLC with FA-SA could further enhance the cytotoxicities of drug in drug sensitive and drug resistant cells.     

功劳木中异汉防己碱对P-糖蛋白介导的人乳腺癌细胞多药耐药性的逆转作用

本文探讨了异汉防己碱对P-糖蛋白(P-gp)介导的人乳腺癌细胞多药耐药性的逆转作用。首先以RT-PCR和免疫组化方法分别从RNA和蛋白水平检测MCF-7/DOX细胞P-gp表达情况，以明确MCF-7/DOX细胞的耐药特征；然后采用MTT法检测异汉防己碱的内在细胞毒性及其对阿霉素(DOX)的增敏作用，并以RF(reversal fold)值评价其逆转效果；同时应用流式细胞仪(FCM)对细胞内DOX的蓄积量进行了分析；再以免疫组化方法检测异汉防己碱对MCF-7/DOX细胞P-gp表达水平的影响；最后采用罗丹明蓄积和外排试验检测了异汉防己碱对P-gp功能的影响。整个试验以维拉帕米作为阳性对照。实验结果表明：MCF-7/DOX细胞是具有多药耐药表型且P-gp表达阳性的细胞株；无毒剂量异汉防己碱可明显增强DOX对MCF-7/DOX细胞的细胞毒性(RF=3.89)，明显高于维拉帕米(RF=2.54)的逆转活性(P<0.05)，但其几乎不影响DOX对MCF-7细胞的抑制作用；异汉防己碱对MCF-7/DOX细胞P-gp表达水平无明显影响，但其可有效抑制P-gp的药物外排功能。因此，异汉防己碱可有效逆转P-gp介导的人乳腺癌细胞的多药耐药性，它可能成为有效多药耐药逆转剂的候选药物。     

Dihydroartemisinin and doxorubicin co-loaded Soluplus®-TPGS mixed micelles: formulation characterization,cellular uptake,and pharmacodynamic studies

Clinically, co-delivery of chemotherapeutics has been limited by poor water-solubility and severe systemic toxicity. This study was aimed at integrating the merits of combination chemotherapy and mixed micellar technology and demonstrating the anticancer potential of doxorubicin (DOX) and dihydroartemisinin (DHA) co-loaded Soluplus^®-TPGS mixed micellar system. In this study, physiochemically stable multidrug loaded mixed micelles were successfully prepared, encapsulation efficiencies of DOX and DHA were as high as 90%, and the average diameter of the micelles was 64.27?nm. The cellular uptake of DOX from the mixed micelles increased by 1.3 and 1.2 times for MCF-7 and MCF-7/ADR cell lines, respectively. The micelles were more cytotoxic than free DHA–DOX. Surprisingly, the co-loaded mixed micelles exhibited higher antitumor activity, while the systemic toxicity was reduced during the treatment. Therefore, the DOX and DHA mixed micelle might be a potential, effective, and less toxic drug-delivery system for cancer therapy.     

pH and Redox Dual Responsive Nanoparticle for Nuclear Targeted Drug Delivery

To mimic the clinic dosing pattern, initially administering high loading dose and then low maintenance dose, we designed a novel poly(2-(pyridin-2-yldisulfanyl)ethyl acrylate) (PDS) based nanoparticle delivery system. Side chain functional PDS was synthesized by free radical polymerization. Polyethylene glycol and cyclo(Arg-Gly-Asp-d-Phe-Cys) (cRGD) peptide was conjugated to PDS through thiol-disulfide exchange reaction to achieve RPDSG polymer. RPDSG/DOX, RPDSG nanoparticle loaded with doxorubicin, was fabricated by cosolvent dialysis method. The size of the nanoparticles was 50.13 ± 0.5 nm in PBS. The RPDSG/DOX nanoparticle is stable in physiological condition while quickly releasing doxorubicin with the trigger of acidic pH and redox potential. Furthermore, it shows a two-phase release kinetics, providing both loading dose and maintenance dose for cancer therapy. The conjugation of RGD peptide enhanced the cellular uptake and nuclear localization of the RPDSG/DOX nanoparticles. RPDSG/DOX exhibits IC(50) close to that of free doxorubicin for HCT-116 colon cancer cells. Due to the synergetic effect of RGD targeting effect and its two-phase release kinetics, RPDSG/DOX nanoparticles display significantly higher anticancer efficacy than that of free DOX at concentrations higher than 5 μM. These results suggest that RPDSG/DOX could be a promising nanotherapeutic for tumor-targeted chemotherapy.     

Cytotoxic and Antitumor Activities of Doxorubicin Conjugates with the Epidermal Growth Factor and its Receptor-binding Fragment

The epidermal growth factor (EGF) receptor is expressed at high levels on many types of tumor cells, such as squamous carcinoma, breast cancer and endothelial cells. We studied targeted delivery of the anticancer drug doxorubicin (DOX) using EGF and its receptor-binding fragment (EGFfr) to cells able to overexpress EGF receptors. EGF-DOX and EGFfr-DOX conjugates were synthesized via a glutaraldehyde bridge. The cytotoxic activities (CTA) of the conjugates were studied in vitro in different tumor cell lines (MCF-7 Wt, MCF-7 AdrR, B16) and endothelial cells using MTT-test. The antitumor effects of the conjugates were examined in vivo in mice with a subcutaneous B16 model. In the case of MCF-7 Wt cells, CTA of EGF-DOX and EGFfr-DOX conjugates exceeded 7.7- and 68-fold that of free DOX. Besides, the conjugates effectively decreased the drug resistance of MCF-7 AdrR cells. CTA of the conjugates against endothelial cell cultures markedly exceeded that of free DOX. It is of note that proliferating endothelial cells were much more sensitive to the effects of the conjugates than confluent endothelial cells. Administration of EGF-DOX and EGFfr-DOX conjugates significantly inhibited tumor growth and increased the mean life span of experimental animals by 46 and 48.5%, respectively.     

Cytotoxic and antitumor activities of doxorubicin conjugates with the epidermal growth factor and its receptor-binding fragment

The epidermal growth factor (EGF) receptor is expressed at high levels on many types of tumor cells, such as squamous carcinoma, breast cancer and endothelial cells. We studied targeted delivery of the anticancer drug doxorubicin (DOX) using EGF and its receptor-binding fragment (EGFfr) to cells able to overexpress EGF receptors. EGF-DOX and EGFfr-DOX conjugates were synthesized via a glutaraldehyde bridge. The cytotoxic activities (CTA) of the conjugates were studied in vitro in different tumor cell lines (MCF-7Wt, MCF-7AdrR, B16) and endothelial cells using MTT-test. The antitumor effects of the conjugates were examined in vivo in mice with a subcutaneous B16 model. In the case of MCF-7Wt cells, CTA of EGF-DOX and EGFfr-DOX conjugates exceeded 7.7- and 68-fold that of free DOX. Besides, the conjugates effectively decreased the drug resistance of MCF-7AdrR cells. CTA of the conjugates against endothelial cell cultures markedly exceeded that of free DOX. It is of note that proliferating endothelial cells were much more sensitive to the effects of the conjugates than confluent endothelial cells. Administration of EGF-DOX and EGFfr-DOX conjugates significantly inhibited tumor growth and increased the mean life span of experimental animals by 46 and 48.5%, respectively.     

A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drug targeting

One of the new strategies to improve cancer chemotherapy is based on new drug delivery systems, like the polyethylene glycol-coated superparamagnetic iron oxide nanoparticles (PEG-SPION, thereafter called PS). In this study, PS are loaded with doxorubicin (DOX) anticancer drug, using a pre-formed DOX-Fe²⁺ complex reversible at lower pH of tumour tissues and cancer cells. The DOX loaded PS (DLPS, 3% w/w DOX/iron oxide) present a hydrodynamic size around 60 nm and a zeta potential near zero at physiological pH, both parameters being favourable for increased colloidal stability in biological media and decreased elimination by the immune system. At physiological pH of 7.4, 60% of the loaded drug is gradually released from the DLPS in ∼2 h. The intracellular release and distribution of DOX is followed by means of confocal spectral imaging (CSI) of the drug fluorescence. The in vitro cytotoxicity of the DLPS on MCF-7 breast cancer cells is equivalent to that of a DOX solution. The reversible association of DOX to the SPION surface and the role of polymer coating on the drug loading/release are discussed, both being critical for the design of novel stealth magnetic nanovectors for chemotherapy.     

载多柔比星二氧化钛纳米粒的制备及体外评价

目的制备载多柔比星（doxorubicin,DOX）的二氧化钛（Ti02）纳米粒,并考察其体外释放百分率及细胞毒性。方法通过水热法合成DOX的Ti02纳米粒,采用透射电镜及X-射线衍射仪对其进行表征,紫外可见分光光度法测定载药量及体外释放,采用MTT法分析其对MCF-7细胞和Hela细胞的细胞毒性。结果所制备的纳米粒分散均匀。外观呈梭状,长度约为200nm,在水中的载药量达10．85％,体外释放具有pH敏感性,空白纳米粒细胞毒性较低,载药纳米粒的细胞毒性与游离多柔比星相当。结论所制备的TiO2纳米粒具有较高的载药量及pH敏感的体外释放性能,可作为DOX的载体。     

Pegylated phosphotidylethanolamine inhibiting P-glycoprotein expression and enhancing retention of doxorubicin in MCF7/ADR cells

The failure of the clinical treatment of cancer patients is often attributed to drug resistance of the tumor to chemotherapeutic agents. P-glycoprotein (P-gp) contributes to drug resistance via adenosine 5'-triphosphate (ATP)-dependent drug efflux pumps and is widely expressed in many human cancers. Up to date, a few of nanomaterials have shown the effects on P-gp function by different ways. To study the mechanism of the increased cytotoxicity of doxorubicin (DOX) by pegylated phosphotidylethanolamine (PEG-PE) in drug-resistant cancer cells, a series of in vitro cell assays were performed, including identification of P-gp function, quantitative studies on uptake and efflux of DOX, inhibitory effects of blank PEG-PE micelles on mRNA and protein levels of P-gp, and intracellular ATP content alteration. Finally, combining MDR-1 RNA interference (siRNA) with DOX encapsulated in PEG-PE micelles (M-DOX) to improve cytotoxicity of DOX was also studied. M-DOX showed fivefold lower the concentration that caused 50% killing tumor cell than that of free DOX in the P-gp-overexpressing MCF-7 breast cancer (MCF-7/ADR) cells. M-DOX enhanced the cellular uptake and retention of DOX in MCF-7/ADR cells. PEG-PE block molecules can inhibit P-gp expression through downregulating MDR-1 gene. Cytotoxicity of M-DOX was further improved by knocking down the MDR-1 gene using siRNA in the multidrug-resistant cells. We conclude that the increased cytotoxicity of DOX encapsulated in PEG-PE micelle is due to the reduced P-gp expression by PEG-PE block molecules, and accordingly enhancing the cellular accumulation of DOX. To overcome drug resistance of tumor cells, the combination of nanotechnology and biotechnology could be an effective strategy such as PEG-PE formed micelles and siRNA.     

Naringenin Enhances the Anti-Tumor Effect of Doxorubicin Through Selectively Inhibiting the Activity of Multidrug Resistance-Associated Proteins but not P-glycoprotein

Purpose  Naringenin has shown paradoxical results to modulate the function of multidrug resistance-associated proteins (MRPs). The aim of this study is to interpret whether naringenin can reverse intrinsic and/or acquired resistance of cancer cells to chemotherapeutic agents. Methods  The effects of naringenin on the uptake, retention and cytotoxicity of doxorubicin were investigated in A549, MCF-7, HepG2 and MCF-7/DOX cells. Cellular efflux pathways modulated by naringenin were assessed with their specific substrates and inhibitors. The improved antitumor activity of doxorubicin in combination with naringenin was also investigated in vivo. Results  The IC₅₀ values of doxorubicin in combination with naringenin in A549 and MCF-7 cells were approximately 2-fold lower than that of doxorubicin alone. The increased sensitivity to doxorubicin by naringenin in HepG2 and MCF-7/DOX cells was not observed. Naringenin increased the cellular doxorubicin accumulation through inhibiting doxorubicin efflux in the cells expressing MRPs but not P-gp. In contrast to doxorubicin alone, doxorubicin in combination with naringenin enhanced antitumor activity in vivo with low systemic toxicity. Conclusion  Naringenin enhances antitumor effect of doxorubicin by selective modulating drug efflux pathways. Naringenin will be a useful adjunct to improve the effectiveness of chemotherapeutic agents in treatment of human cancers.     

Nuclear targeting and nuclear retention of anthracycline-formaldehyde conjugates implicates DNA covalent bonding in the cytotoxic mechanism of anthracyclines.

The anthracycline, antitumor drugs doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) catalyze production of formaldehyde through induction of oxidative stress. The formaldehyde then mediates covalent bonding of the drugs to DNA. Synthetic formaldehyde conjugates of DOX, DAU, and EPI, denoted Doxoform (DOXF), Daunoform (DAUF), and Epidoxoform (EPIF), exhibit enhanced toxicity to anthracycline-sensitive and -resistant tumor cells. Uptake and retention of parent anthracycline antitumor drugs (DOX, DAU, and EPI) relative to those of their formaldehyde conjugates (DOXF, DAUF, and EPIF) were assessed by flow cytometry in both drug-sensitive MCF-7 cells and drug-resistant MCF-7/ADR cells. The MCF-7 cells took up more than twice as much drug as the MCF-7/ADR cells, and both cell lines took up substantially more of the formaldehyde conjugates than the parent drugs. Both MCF-7 and MCF-7/ADR cells retained fluorophore from DOXF, DAUF, and EPIF hours after drug removal, while both cell lines almost completely expelled DOX, DAU, and EPI within 1 h. Longer treatment with DOX, DAU, and EPI resulted in modest drug retention in MCF-7 cells following drug removal but poor retention of DOX, DAU, and EPI in MCF-7/ADR cells. Fluorescence microscopy showed that the formaldehyde conjugates targeted the nuclei of both sensitive and resistant cells, and remained in the nucleus hours after drug removal. Experiments in which [(3)H]Doxoform was used, synthesized from doxorubicin and [(3)H]formaldehyde, also indicated that Doxoform targeted the nucleus. Elevated levels of (3)H were observed in DNA isolated from [(3)H]Doxoform-treated MCF-7 and MCF-7/ADR cells relative to controls. The results implicate drug-DNA covalent bonding in the tumor cell toxicity mechanism of these anthracyclines.