Reversal of doxorubicin resistance in breast cancer cells by photochemical internalization

Multiple drug resistance (MDR) is a problem that seriously reduces the efficacy of many chemotherapy agents. One mechanism for MDR is increased acidification of endocytic vesicles and increased cytosol pH, so weak base chemotherapeutic agents, including doxorubicin, are trapped in endocytic vesicles and exhibit a drug resistant phenotype. Treatments that selectively reverse this accumulation may therefore reverse the MDR phenotype. Photochemical internalization (PCI) is a novel technology developed for site-specific enhancement of the therapeutic efficacy of macromolecules by selective photochemical rupture of endocytic vesicles and consequent release of endocytosed macromolecules into the cytosol. This study evaluates PCI for release of doxorubicin from endocytic vesicles in MDR cells. Two breast cancer cell lines, MCF-7 and MCF-7/ADR (the latter resistant to doxorubicin), were selected. They were found equally sensitive to photochemical treatment with the photosensitiser TPPS(2) (a) (disulfonated meso-tetraphenylporphine) and light. On exposure to doxorubicin alone, the IC(50) (drug concentration for 50% reduction in colony formation) was 0.1 microM for MCF-7 and 1 microM for MCF-7/ADR. After PCI (photochemical treatment followed by doxorubicin), the IC(50) concentration was 0.1 microM for both cell lines. Comparable changes were seen with assay of cell viability using 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). On fluorescence microscopy in MCF-7/ADR cells, doxorubicin localised in granules identified as lysosomes. After PCI, doxorubicin was released into the cytosol and entered cell nuclei, as was seen in MCF-7 cells without PCI. In conclusion, PCI reversed the MDR phenotype of doxorubicin resistant breast cancer cells by endo-lysosomal release of the drug. The technique is a promising new approach to tackling the problem of MDR.     

Targeted exosome‐encapsulated erastin induced ferroptosis in triple negative breast cancer cells

Ferroptosis is an iron‐dependent, lipid peroxide‐driven cell death caused by inhibition of the cystine/glutamate transporter, which is of importance for the survival of triple‐negative breast cancer (TNBC) cells. Erastin is a low molecular weight chemotherapy drug that induces ferroptosis; however, poor water solubility and renal toxicity have limited its application. Exosomes, as drug delivery vehicles with low immunogenicity, high biocompatibility and high efficiency, have attracted increasing attention in recent years. Herein, we developed a formulation of erastin‐loaded exosomes labeled with folate (FA) to form FA‐vectorized exosomes loaded with erastin (erastin@FA‐exo) to target TNBC cells with overexpression of FA receptors. The characterization, drug release, internalization and anti–tumor effect in vitro of erastin@FA‐exo were determined. Erastin@FA‐exo could increase the uptake efficiency of erastin into MDA‐MB‐231 cells; compared with erastin@exo and free erastin, erastin@FA‐exo has a better inhibitory effect on the proliferation and migration of MDA‐MB‐231 cells. Furthermore, erastin@FA‐exo promoted ferroptosis with intracellular depletion of glutathione and reactive oxygen species overgeneration. Western blot analyses revealed that erastin@FA‐exo suppressed expression of glutathione peroxidase 4 (GPX4) and upregulated expression of cysteine dioxygenase (CDO1). We conclude that targeting and biocompatibility of exosome‐based erastin preparations provide an innovative and powerful delivery platform for anti–cancer therapy.     

乳腺癌多药耐药DEDD作用及机制研究

目的 死亡效应结构域DNA结合蛋白(death effector domain DNA-binging protein,DEDD)可抑制乳腺癌的生长和转移,但在乳腺癌多药耐药中的作用尚不明确.本研究将探讨DEDD在人乳腺癌多药耐药中的作用及机制.方法 通过转染DEDD-shRNA建立稳定敲低DEDD表达的MCF-7人乳腺癌细胞株,MTS实验检测多柔比星、紫杉醇对MCF-7 WT(Wild type,野生型)、MCF-7 control shRNA和MCF-7 DEDD-shRNA细胞活力的影响,Annexin-V和碘化丙啶(propidium iodide,PI)双染后流式细胞仪检测细胞凋亡.蛋白质印迹法检测乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)在药物干预后的蛋白表达水平.结果 多柔比星干预后,半数抑制浓度IC50的多因素方差分析结果显示,不同处理(F=89.49,P＜0.001)、不同时间(F=50.81,P＜0.001)组间比较差异有统计学意义.多柔比星干预24或48 h后,MCF-7 DEDD-shRNA的半数抑制浓度IC50分别为(0.97±0.15)和(0.62±0.09) μmol/L,明显高于MCF-7 WT(0.46±0.05)和(0.26±0.03) μmol/L和MCF-7 control shRNA细胞(0.39±0.05)和(0.25±0.03)μmol/L,P＜0.001.紫杉醇干预后,半数抑制浓度IC50的多因素方差分析结果显示,不同处理(F=15.94,P＜0.001)、不同时间组间(F=129.70,P＜0.001)比较差异有统计学意义.紫杉醇干预24或48 h后,MCF-7 DEDD-shRNA的半数抑制浓度IC50为(16.74±2.26)和(9.88±1.47) μmol/L,明显高于MCF-7 WT(13.39±1.44)和(7.69±0.92) μmol/L(P=0.001)和MCF-7 control shRNA细胞(12.58±1.15)和(7.17±1.12) μmol/L(P＜0.001).多柔比星干预后24 h后流式细胞凋亡结果显示,不同处理(F=131.46,P＜0.001)、不同浓度组间(F=160.95,P＜0.001)比较差异有统计学意义.正常培养状态下,MCF-7 control shRNA细胞和MCF-7 DEDD-shRNA细胞的凋亡率分别为(14.32±1.47)％和(11.58±1.63)％,而给予0.5μmol/L和1 μmol/L多柔比星作用24 h后,M[CF-7DEDD-shRNA细胞的凋亡率分别为(21.62±1.97)％和(24.39±2.36)％,明显低于MCF-7 control shRNA细的(36.26±1.87)％和(38.23±1.46)％,P＜0.001.同样,紫杉醇干预后24 h,不同处理(F=124.81,P＜0.001)、不同浓度组问(F=172.56,P＜0.001)细胞凋亡率比较差异有统计学意义.给予9.37和18.74 μmol/L紫杉醇作用24 h后,MCF-7DEDD-shRNA细胞的凋亡率分别为(30.26±1.63)％和(32.18±2.16)％,明显低于MCF-7 control shRNA组的(53.84±2.17)％和(58.27±2.16)％,P＜0.001.多柔比星作用后不同时间BCRP蛋白质印迹检测统计分析结果显示,不同处理(F=14.67,P＜0.001)、不同时间(F=6.39,P=0.006)组间BCRP蛋白表达比较差异有统计学意义.给予0.5 μmol/L多柔比星作用24 h后,3组细胞的BCRP相对表达水平分别为0.87±0.04、1.06±0.02和5.25±0.18.MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.多柔比星作用48 h后,3组细胞的BCRP相对表达水平分别为1.06±0.01、0.97±0.04和2.98±0.13.MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平出现回落,但仍显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.同样,紫杉醇作用后不同时间BCRP蛋白质印迹检测统计分析结果显示,不同处理(F=7.26,P=0.004)、不同时间(F=8.32,P=0.002)组间BCRP蛋白表达比较差异有统计学意义.给予9.37 μmol/L紫杉醇作用24 h后,3组细胞的BCRP相对表达水平分别为0.81±0.05、2.25±0.10和1.78±0.14.其中MCF-7 control shRNA组细胞BCRP表达水平高于MCF-7 WT (P＜0.001)和MCF-7 DEDD-shRNA (P=0.002)组.而紫杉醇作用48 h后,3组细胞的BCRP相对表达水平分别为0.73±0.04、1.73±0.05和3.12±0.12,MCF-7 DEDD-shRNA细胞组BCRP蛋白的表达水平继续增高,且显著高于MCF-7 WT和MCF-7 control shRNA细胞组,P＜0.001.结论 敲低DEDD表达可增强乳腺癌细胞的抗肿瘤药耐药性,BCRP的表达增加可能是低表达DEDD乳腺癌细胞多药耐药的机制之一.     

乳腺癌耐药蛋白的研究进展

乳腺癌耐药蛋白(BVRP)是新近发现的一种肿瘤耐药相关蛋白．与P-gp和多药耐药相关蛋白(MRP)同属ABC转运蛋白超家族。本文对BCRP的发现、基因表达特征、与造血干细胞分化的关系、转运底物及其耐药逆转和临床意义等方面的研究进展进行综述。     

Heterogeneity ofin vitro chemosensitivity in perioperative breast cancer cells to mitoxantroneversus doxorubicin evaluated by a microplate ATP bioluminescence assay

Summary Apart from clinical trials, mitoxantrone (MX) is rarely used in breast cancer (BC) due to the anticipated anthracycline cross-resistance. We have examined this drug versus doxorubicin (DOX) using data obtained fromin vitro microplate ATP tumor chemosensitivity assays (ATP-TCA) of BC cells which were derived from 55 chemotherapy-naive patients at time of primary surgery. Both drugs were tested at 6 different concentrations ranging from 6.25% to 200% peak plasma concentrationin vivo (PPC). Differences between DOX and MX observed for mean IC₅₀, IC₉₀, and a sensitivity index (SI) were not statistically significant.In vitro response rates were 44% for DOX and 52% for MX. 34 of 52 eligible assays (65%) showed comparable activity of both drugs whereas a lack of cross-resistance was observed in the remaining 18 (35%) tumors as indicated by differences for SI. Cumulative concentration-response plots of tumors respondingin vitro with a 50 percent or 90 percent tumor cell inhibition showed a strong dose-dependence for both DOX and MX at concentrations which normally can be achieved within clinical tumors (i.e. 6.25%-50% PPC). At higher concentrations, however, cytotoxicity of DOX and MX could not be improved by furtherin vitro dose escalation. Moreover, a substantial proportion of BC specimens (DOX: 48.1%; MX: 40.4%) did not experience a 90 tumor cell inhibition at 200% PPC. In conclusion,in vitro results obtained by ATP-TCA indicate that there is no cross-resistance between MX and DOX in a substantial proportion of BC patients. This may be clinically useful and suggests that combinations including MX should be tested in patients clinically resistant to DOX containing regimens. Since both drugs produced sigmoidal concentration-response curves, dose escalation beyond a certain point may not produce increased sensitivity.     

Reversal effects of Raloxifene on paclitaxel resistance in 2 MDR breast cancer cells

Raloxifene hydrochloride (RAL), one of second generation of selective estrogen receptor modulators (SERMs), is usually used in preventing osteoporosis and breast cancer. The present study evaluated whether Raloxifene might sensitize multidrug resistant (MDR) breast cancers to chemotherapies, especially in estrogen receptor negative (ER−) breast cancer. The results showed that RAL could significantly sensitize ER- MDR breast tumors to paclitaxel both in vitro and in vivo. Combination of Raloxifene could significantly enhance paclitaxel-induced cell apoptosis, G2-M arrest as well as inhibition of cell proliferation in MDR tumors. Further studies showed that the combined treatment did not alter P-glycoprotein expression but increased P-gp ATPase activity. These results suggested that raloxifene might be a valuable chemosensitizer agent for breast cancer therapy.     

Uptake and distribution of doxorubicin in hormone-manipulated human breast cancer cells in vitro

Background: Kinetic resistance to cytotoxic drugs may account for the moderate responsiveness of breast cancer to chemotherapy. In the present study the in vitro effects of estradiol-mediated DNA stimulation on the cellular uptake of the DNA intercalating drug doxorubicin (DOX) were examined in MCF-7 human breast cancer cells. Methods: Using the fluorescent properties of the drug, the cellular uptake was investigated by high performance liquid chromatography (HPLC), and by flow cytometry. Results: The uptake of DOX (0.01–2 g/ml) by MCF-7 cells in suspension, incubated for 1 and 6 h, showed a strong correlation between the incubation concentration of DOX and the cellular uptake of the drug as measured by HPLC and flow cytometry. Simultaneous exposure of MCF-7 cells, in monolayer culture, to DOX (0.04–0.2 g/ml) and estradiol (1 nM) for 1–24 h showed no significant difference in uptake of the drug compared to control cultures exposed to DOX in the absence of estradiol. Neither was there a significant difference in uptake of DOX when MCF-7 cells were pretreated with estradiol (1 nM) for 16–24 h followed by a 0.5, 1, 6, and 21/23 h incubation with DOX (0.01–2 g/ml). Pretreatment with estradiol did not affect the retention of DOX as measured 24 h after a 0.5 h incubation with DOX (2 g/ml). Furthermore, fluorescence microscopy revealed no difference in the cellular DOX distribution pattern of estradiol-stimulated MCF-7 cultures compared to unstimulated cultures. Conclusion: From this study we can conclude that, for the human MCF-7 breast cancer cells in vitro, the uptake, retention, and cellular distribution of DOX are not influenced by estrogenic manipulation.     

双特异性溶瘤腺病毒和多柔比星对乳腺癌细胞增殖抑制作用的对比分析

目的:探讨多柔比星和双特异性溶瘤腺病毒（Ad-VT、Ad-T、Ad-VP3、Ad-Mock）对乳腺癌细胞和正常乳腺细胞增殖抑制作用的差异。方法：通过WST-1 实验比较多柔比星和Ad-VT、Ad-T、Ad-VP3、Ad-Mock对乳腺癌细胞增殖的抑制率,并比较两种药物对正常乳腺上皮细胞的存活率的影响。通过Annexin V流式术、Hoechst 法、JC-1 法检测研究溶瘤腺病毒和多柔比星对乳腺癌细胞和正常乳腺上皮细胞杀伤作用的影响,并比较其凋亡率差异。结果：4 种双特异性溶瘤腺病毒均能有效地抑制乳腺癌细胞的增殖（P<0.05 或P<0.01）,且抑制效果为Ad-VT>Ad-T>Ad-VP3>Ad-MOCK,抑制作用与时间成正相关。多柔比星也能有效地抑制乳腺癌细胞的增殖（P<0.05 或P<0.01）,且随着浓度和时间的增加,抑制效果明显增强;但多柔比星对正常的乳腺上皮细胞也有较强的抑制作用,在72 h、5 μg/ml 条件下抑制率达到80%,而溶瘤腺病毒Ad-VT在72 h 对MCF-10A的抑制率为20%。双特异性溶瘤腺病毒诱导乳腺癌凋亡能力随时间的增加逐渐增强（P<0.05 或P<0.01）,且致凋亡效率为Ad-VT>Ad-T>Ad-VP3>Ad-MOCK,而诱导正常乳腺细胞凋亡能力较弱。多柔比星诱导乳腺癌细胞和正常乳腺上皮细胞凋亡的能力基本相同（P<0.05 或P<0.01）,且都为0.05<0.5<5 μg/ml。结论：双特异性溶瘤腺病毒能有效地抑制乳腺癌细胞的增殖,但对正常的乳腺细胞抑制作用较小,双特异性溶瘤腺病毒具有更好的安全性,为肿瘤的生物治疗提供了新的药物。     

乳腺癌上皮-间质转化后引发BCRP介导的多药耐药的研究

背景与目的:研究发现肿瘤的侵袭浸润和转移增强是上皮-间质转化(epithelial mesenchymal transition, EMT)所致,而EMT的发生也与肿瘤细胞多药耐药现象的发生密切相关.本研究通过分析乳腺癌组织和细胞中EMT与乳腺癌耐药蛋白表达的相关性,探讨EMT对乳腺癌中乳腺癌耐药蛋白(breast cancer resistant protein, BCRP)介导多药耐药的影响.方法:免疫组织化学方法检测乳腺癌组织中Snail、BCRP的表达:构建Snail真核表达载体pCDNA3.1-Snail,转染至MCF-7细胞,采用免疫荧光、Western blot、Real-time PCR检测转录抑制因子snail、上皮标志物E-钙粘素、间质标志物vimentin以及多药耐药蛋白BCRP的表达;MTT法检测细胞对米托蒽醌的耐药指数.结果:免疫组化结果显示乳腺癌组织中Snail、BCRP的表达呈显著相关:免疫荧光、Western blot、Real-time PCR显示与亲本MCF-7细胞相比,转染Snail后的MCF-7细胞中E-cadherin表达水平明显降低,而snail、vimentin和BCRP的表达水平明显增加;MTT结果显示细胞对米托蒽醌的耐药指数增加至9.93.结论:转染snail真核表达载体pCDNA3.1-snail可使乳腺癌MCF-7细胞发生EMT, 并导致细胞中BCRP表达增加,引发BCRP介导的多药耐药.     

DHA在人乳腺癌中对阿霉素细胞毒活性的影响与脂质过氧化作用的关系

目的　探讨廿二碳六烯酸 (DHA)影响阿霉素 (ADM)对人乳腺癌DMA MB 435s细胞毒活性的机理是否通过DHA的脂质过氧化作用。方法　在培养的人乳腺癌DMA MB 435s细胞株中加入不同配伍的细胞毒药物、多不饱和脂肪酸 (PUFAs)、前氧化剂VitC和VitK3混合物及抗氧化剂VitE等 ,在细胞的抽提物中以TBA法每 2 4小时测定脂质过氧化产物丙二醛 (MDA)及用亚硝酸盐分光光度法测定一氧化氮 (NO)的含量 ,并作出MDA、NO含量与细胞毒性间的剂量 -效应相关直线。结果　从细胞培养的第三天开始 ,出现明显的细胞活力变化 ,同时伴有脂质过氧化物 (MDA)水平的增加及NO含量的降低。细胞抽提物中MDA及NO含量与细胞毒性之间存在直线相关关系。结论　DHA能明显增加ADM对MDA MB 435s细胞株的细胞毒活性 ,机理之一是DHA增强了瘤细胞内的脂质过氧化作用。     

Sequence- and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells

We investigated whether the combination of zoledronic acid and doxorubicin induced apoptosis of breast and prostate cancer cell lines, and if synergistic interaction was present. We investigated whether the levels of cell death altered depending on the sequence in which the drugs were administered and the possible mechanism of action responsible for the increased cell death following combined treatments. Breast and prostate cancer cells were treated with zoledronic acid alone, doxorubicin alone, or drugs in sequence (doxorubicin before, after, or with zoledronic acid), and the levels of apoptotic death were determined by evaluation of nuclear morphology. We found that clinically relevant concentrations of doxorubicin and zoledronic acid induced sequence- and schedule-dependent apoptosis of breast and prostate cancer cells. For maximal apoptosis, cells had to be pretreated for 24 hr with doxorubicin before immediate treatment with zoledronic acid for 1 hr. This observation is a characteristic feature of cell cycle phase-specific synergistic effect. Replacing zoledronic acid with the nonnitrogen-containing bisphosphonate clodronate did not induce increased apoptosis. Induction of apoptosis was mainly via inhibition of the mevalonate (MVA) pathway, as addition of the MVA pathway intermediary geranylgeraniol inhibited the induction of apoptosis by doxorubicin followed by zoledronic acid. In conclusion, combined treatment of breast and prostate cancer cell lines with clinically relevant doses of doxorubicin and zoledronic acid induces apoptosis in a synergistic fashion. These findings may have relevance for the clinical setting, particularly breast cancer patients receiving these drugs in the adjuvant setting.     

A leukotriene B4 receptor-2 is associated with paclitaxel resistance in MCF-7/DOX breast cancer cells

Background:

Breast cancer is the most common malignancy in women. Although chemotherapeutic agents, such as paclitaxel, are effective treatments for the majority of breast cancer patients, recurrence is frequent and often leads to death. Thus, there is an urgent need to identify novel therapeutic targets that sensitise tumour cells to existing chemotherapy agents.

Methods:

The levels of leukotriene B₄ receptor-2 (BLT2) in multidrug-resistant MCF-7/DOX cells were determined using quantitative PCR and FACS analysis. The potential role of BLT2 in the paclitaxel resistance of MCF-7/DOX cells was assessed using a pharmacological inhibitor and small interfering RNA knockdown, and the BLT2-associated resistance mechanism was assessed.

Results:

The expression levels of BLT2 were markedly upregulated in MCF-7/DOX cells. The inhibition of BLT2 by pre-treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283 or siBLT2 knockdown significantly sensitised MCF-7/DOX cells to paclitaxel and induced significant levels of apoptotic death, suggesting that BLT2 mediates paclitaxel resistance. We also demonstrated that BLT2-induced paclitaxel resistance was associated with the upregulation of P-glycoprotein. Finally, co-treatment with a BLT2 inhibitor and paclitaxel markedly reduced tumour growth in an MCF-7/DOX in vivo model.

Conclusion:

Together, our results demonstrate that BLT2 is a novel therapeutic target that sensitises drug-resistant breast cancer cells to paclitaxel.     

Indole‐3‐carbinol inhibits MDA‐MB‐231 breast cancer cell motility and induces stress fibers and focal adhesion formation by activation of Rho kinase activity

Indole‐3‐carbinol (I3C), a phytochemical derived from cruciferous vegetables such as broccoli and Brussels sprouts, has potent antiproliferative effects in human breast cancer cells and has been shown to decrease metastatic spread of tumors in experimental animals. Using chemotaxis and fluorescent‐bead cell motility assays, we demonstrated that I3C significantly decreased the in vitro migration of MDA‐MB‐231 cells, a highly invasive breast cancer cell line. Immunofluorescence staining of the actin cytoskeleton revealed that concurrent with the loss of cell motility, I3C treatment significantly increased stress fiber formation. Furthermore, I3C induced the localization of the focal adhesion component vinculin and tyrosine‐phosphorylated proteins to the cell periphery, which implicates an indole‐dependent enhancement of focal adhesions within the outer boundary of the cells. Coimmunoprecipitation analysis of focal adhesion kinase demonstrated that I3C stimulated the dynamic formation of the focal adhesion protein complex without altering the total level of individual focal adhesion proteins. The RhoA‐Rho kinase pathway is involved in stress fiber and focal adhesion formation, and I3C treatment stimulated Rho kinase enzymatic activity and cofilin phosphorylation, which is a downstream target of Rho kinase signaling, but did not increase the level of active GTP‐bound RhoA. Exposure of MDA‐MB‐231 cells to the Rho kinase inhibitor Y‐27632, or expression of dominant negative RhoA ablated the I3C induced formation of stress fibers and of peripheral focal adhesions. Expression of constitutively active RhoA mimicked the I3C effects on both processes. Taken together, our data demonstrate that I3C induces stress fibers and peripheral focal adhesions in a Rho kinase‐dependent manner that leads to an inhibition of motility in human breast cancer cells. © 2008 Wiley‐Liss, Inc.