Targeting autophagy in breast cancer

Macroautophagy (referred to as autophagy here) is an intracellular degradation pathway enhanced in response to a variety of stresses and in response to nutrient deprivation. This process provides the cell with nutrients and energy by degrading aggregated and damaged proteins as well as compromised organelles. Since autophagy has been linked to diverse diseases including cancer, it has recently become a very interesting target in breast cancer treatment. Indeed, current clinical trials are trying to use chloroquine or hydroxychloroquine, alone or in combination with other drugs to inhibit autophagy during breast cancer therapy since chemotherapy and radiation, regimens that are used to treat breast cancer, are known to induce autophagy in cancer cells. Importantly, in breast cancer, autophagy has been involved in the development of resistance to chemotherapy and to anti-estrogens. Moreover, a close relationship has recently been described between autophagy and the HER2 receptor. Here, we discuss some of the recent findings relating autophagy and cancer with a particular focus on breast cancer therapy.     

肿瘤相关成纤维细胞自噬对三阴性乳腺癌细胞转移的影响

目的探讨三阴性乳腺癌(TNBC)细胞和肿瘤相关成纤维细胞(CAFs)共培养环境下,CAFs自噬促进TNBC细胞转移的作用机制。 方法(1)分离和鉴定CAFs：采用前瞻性研究方法收集2015年1月至2016年12月期间,就诊于南方医科大学珠江医院的6例TNBC患者(临床分期Ⅱ～Ⅲ期)的肿瘤组织和癌旁正常组织,从这些标本中分离CAFs和正常成纤维细胞(NFs)。进一步采用荧光抗体标记抗α平滑肌肌动蛋白(α-SMA),并计算α-SMA阳性细胞比例,采用Western blot检测CAFs和NFs的α-SMA表达水平,以鉴定CAFs为本实验所需。(2)评估CAFs自噬水平：利用Western blot检测CAFs组、NFs组和CAFs＋3-MA组(加入自噬抑制剂3-MA的CAFs)自噬标志蛋白beclin 1和p62的表达水平。(3)明确CAFs自噬对MDA-MB-231和BT-549细胞迁移的作用：采用Transwell小室模型观察不同培养条件[分4组,包括空白组(含10% FBS的DMEM培养基,即普通培养基)、NFs组、CAFs组和CAFs＋3-MA组]下MDA-MB-231和BT-549的细胞迁移数。(4)明确CAFs自噬对MDA-MB-231和BT-549细胞发生上皮-间质转化(EMT)的影响：采用Western blot检测3种培养条件[普通培养基(空白组)、CAFs条件培养基(CAFs-CM组)和加入3-MA的CAFs条件培养基(CAFs＋3-MA-CM组)]下MDA-MB-231和BT-549 EMT相关蛋白(E-cadherin,N-cadherin, vimentin)的表达情况。正态分布的数据用±s表示,偏态分布的数据用M(P₂₅-P₇₅)表示。2组间α-SMA表达水平比较采用两独立样本非参数秩和检验,α-SMA阳性细胞比例比较采用两独立样本t检验,各组间细胞自噬标志蛋白表达水平、细胞迁移数和EMT相关蛋白表达水平的比较均采用单因素方差分析。 结果(1)荧光显微镜下观察发现,每视野下CAFs组α-SMA阳性细胞比例显著高于NFs组[(81.11±3.95)%比(5.11±2.37)%,t＝49.49, P<0.001); Western blot检测表明,CAFs组α-SMA表达水平也高于NFs组[M(P₂₅～P₇₅)：0.98(0.95～0.98)比0.48(0.47～0.48),Z＝2.023, P＝0.043]。(2)CAFs组、NFs组和CAFs＋3-MA组细胞beclin 1表达水平分别为0.99±0.03、0.73±0.04和0.26±0.02,p62表达水平分别为0.75±0.02、0.98±0.03和0.97±0.01,组间比较,差异均有统计学意义(F＝546.188、136.353,P均<0.001),其中,CAFs组的beclin 1表达水平显著高于CAFs＋3-MA组(P<0.001),p62表达水平明显低于CAFs＋3-MA组(P<0.001)。(3)CAFs组、NFs组、CAFs＋3-MA组以及空白组MDA-MB-231细胞迁移数分别为(41.67±2.78)、(23.33±2.18)、(22.00±1.76)、(18.00±2.12)个,4组比较,差异有统计学意义(F＝198.374,P<0.001);同样,4组BT-549细胞的迁移数分别为(35.22±1.97)、(22.00±2.60)、(25.11±2.15)、(15.22±2.00)个,组间比较,差异也有统计学意义(F＝129.424,P<0.001),并且,CAFs组的MDA-MB-231和BT-549细胞迁移数均明显高于其余3组(P<0.050)。(4)MDA-MB-231细胞分别与普通培养基(空白组)、CAFs条件培养基(CAFs-CM组)和加入3-MA的CAFs条件培养基(CAFs＋3-MA-CM组)共培养后,3组细胞的E-cadherin表达水平分别为0.79±0.03、0.54±0.02和0.87±0.04, N-cadherin表达水平分别为0.59±0.02、1.00±0.02和0.93±0.02, vimentin表达水平分别为0.62±0.03、1.01±0.01和0.89±0.09,组间比较,差异也均有统计学意义(F＝139.286、604.905、43.884,P均<0.001)。同样,BT-549细胞分别与以上3种培养基共培养后,3组细胞的E-cadherin表达水平分别为1.01±0.03、0.63±0.03和0.98±0.03, N-cadherin表达水平分别为0.58±0.01、0.94±0.04和0.95±0.03, vimentin表达水平分别为0.61±0.01、0.98±0.03和0.75±0.02,组间比较,差异也均有统计学意义(F＝210.102、184.477、217.659,P均<0.001)。 结论CAFs可促进TNBC细胞MDA-MB-231和BT-549的迁移和EMT过程,且该作用与CAFs自噬相关。     

放射诱导多倍体乳腺癌细胞衰老和自噬

目的探讨衰老和自噬在放射诱导的多倍体乳腺癌细胞中的作用。方法在6 MV X射线模式下用7 Gy剂量照射乳腺癌MDA⁃MB⁃231细胞,分别于第3天(Day 3组)、5天(Day 5组)、7天(Day 7组)、11天(Day 11组)和19天(Day 19组)观察细胞形态变化,采用流式细胞术检测细胞倍性,β⁃半乳糖苷酶检测细胞衰老情况,Western blot检测衰老和自噬相关蛋白的表达。应用GEPIA工具分析PLK1在乳腺组织及乳腺癌组织中的表达差异。结果7 Gy剂量照射后,Day 3组、Day 5组、Day 7组MDA⁃MB⁃231细胞体积变大,多倍体细胞亚群(DNA含量>4 N)比例均明显高于未经放射处理的对照组MDA⁃MB⁃231细胞(均P<0.0001),同时发生细胞衰老现象。与未经放射处理的对照组相比,Day 3组和Day 5组中DNA损伤修复蛋白PARP,DNA合成相关蛋白Rb、E2F⁃1、E2F⁃2表达下调,自噬相关蛋白LC3B/LC3A比值显著升高;核膜完整性相关蛋白Lamin B1、DNA损伤修复蛋白PARP表达下调,DNA损伤应答相关蛋白PLK1表达上调,差异均有统计学意义(均P<0.05)。与乳腺组织相比,PLK1在乳腺癌组织中高表达(P<0.05)。结论放射诱导乳腺癌细胞发生衰老现象可能是多倍体细胞形成的有利条件,衰老和自噬现象也可能有助于多倍体细胞自我修复去倍化增殖。     

Ev vivo organ culture as potential prioritization tool for breast cancer targeted therapy

The growing use of genomic testing presents new treatment options but also new dilemmas. We describe here a heavily-pretreated metastatic triple negative breast cancer patient who failed to respond to conventional treatment. Genomic analyses were performed that discovered several targetable alterations (e.g. FGFR1, CDK6, INSR) and created a clinical challenge – which target to target first? Our solution to this relatively common scenario was using ex-vivo organ culture (EVOC) system to prioritize treatment directed toward the best molecular target. EVOC enabled the trial of several potent targeted agents (Everolimus, Linsitinib, Palbociclib, AZD4547) and allowed semi-quantitative measurement of tumor response. The best response was to FGFR inhibitor, AZD4547. Consequently, the most accessible FGFR inhibiting agents (Pazopanib, then Nintedanib) were administered and some response was achieved. This report provides a potential rationale for utilizing EVOC system to predict tumor response to targeted therapy when multiple targets are proposed.     

Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells

Ginsenoside F2 (F2) was assessed for its antiproliferative activity against breast cancer stem cells (CSCs). F2 induced apoptosis in breast CSCs by activating the intrinsic apoptotic pathway and mitochondrial dysfunction. Concomitantly, F2 induced the formation of acidic vesicular organelles, recruitment of GFP-LC3-II to autophagosomes, and elevation of Atg-7 levels, suggesting that F2 initiates an autophagic progression in breast CSCs. Treatment with an inhibitor of autophagy enhanced F2-induced cell death. Our findings provide new insights into the anti-cancer activity of F2 and may contribute to the rational use and pharmacological study of F2.     

MicroRNA-25 regulates chemoresistance-associated autophagy in breast cancer cells,a process modulated by the natural autophagy inducer isoliquiritigenin

Recent findings have revealed that dysregulated miRNAs contribute significantly to autophagy and chemoresistance. Pharmacologically targeting autophagy-related miRNAs is a novel strategy to reverse drug resistance. Here, we report a novel function of isoliquiritigenin (ISL) as a natural inhibitor of autophagy-related miR-25 in killing drug-resistant breast cancer cells. ISL induced chemosensitization, cell cycle arrest and autophagy, but not apoptosis, in MCF-7/ADR cells. ISL also promoted the degradation of the ATP-binding cassette (ABC) protein ABCG2 primarily via the autophagy-lysosome pathway. More importantly, miRNA 3.0 array experiments identified miR-25 as the main target of ISL in triggering autophagy flux. A mechanistic study validated that miR-25 inhibition led to autophagic cell death by directly increasing ULK1 expression, an early regulator in the autophagy induction phase. miR-25 overexpression was demonstrated to block ISL-induced autophagy and chemosensitization. Subsequent in vivo experiments showed that ISL had chemosensitizing potency, as revealed by an increase in LC3-II staining, the downregulation of ABCG2, a reduction in miR-25 expression and the activation of the miR-25 target ULK1. Overall, our results not only indicate that ISL acts as a natural autophagy inducer to increase breast cancer chemosensitivity, but also reveal that miR-25 functions as a novel regulator of autophagy by targeting ULK1.     

Promotion of autophagy as a mechanism for radiation sensitization of breast tumor cells

Radiation has long been a useful component of the treatment regimen for solid tumors. However, some malignancies are relatively resistant to radiation treatment while even tumors that may initially respond (to both radiation and chemotherapy) may eventually recover proliferative capacity. A variety of approaches have been utilized in the efforts to enhance radiation sensitivity. Recent studies have identified autophagy as a cell death pathway that may mediate the radiosensitizing effects of selected treatments. Studies in our laboratory support the premise that radiosensitization of breast tumor cells by vitamin D or vitamin D analogs is mediated through autophagy. In addition, promotion of autophagic cell death by a vitamin D analog in irradiated breast tumor cells delays and attenuates the proliferative recovery that may be a preclinical indicator of disease recurrence.     

A novel dithiocarbamate analogue with potentially decreased ALDH inhibition has copper-dependent proteasome-inhibitory and apoptosis-inducing activity in human breast cancer cells

Dithiocarbamates are a class of sulfur-based metal-chelating compounds with various applications in medicine. We reported previously that certain members of dithiocarbamates, such as diethyldithiocarbamate, disulfiram (DSF) and pyrrolidine dithiocarbamate (PDTC), were able to bind with tumor cellular copper to inhibit tumor growth through the inhibition of proteasome activity and induction of cancer cell apoptosis. Since the DSF is an irreversible inhibitor of aldehyde dehydrogenase (ALDH), its ALDH-inhibitory activity might potentially affect its usefulness as an anti-cancer drug. For the purpose of selecting potent anti-cancer compounds that are not ALDH inhibitors and mapping out preliminary structure-activity relationship trends for these novel compounds, we synthesized a series of PDTC analogues and chose three novel compounds to study their ALDH-inhibitory activity, proteasome-inhibitory activity as well as the cancer cell apoptosis-inducing activity. The results showed that compared to DSF, compound 9 has less ALDH inhibition activity, and the in vitro results also proved the positive effects of 9-Cu in proteasome inhibition and apoptosis induction in breast cancer cells, suggesting that 9 as a lead compound could be developed into a novel proteasome inhibitor anti-cancer drug.     

Growth-inhibitory effect of adiponectin via adiponectin receptor 1 on human breast cancer cells through inhibition of S-phase entry without inducing apoptosis

Background The mammographic features of triple receptor-negative [TRN] breast cancers, a distinct cancer subtype with a poor prognosis have not been reported to our knowledge. The aim of this study was to compare the mammographic breast density, visibility, and tumor features of different breast cancer immunophenotypes. Patients and methods We identified all premenopausal women aged 45 years or less who had been diagnosed with primary breast cancer between January 1999 and November 2005 at a single institution and who had undergone mammography at initial diagnosis. Patient characteristics including clinical, histologic, and mammographic features of breast cancers were tabulated by immunophenotype and compared with the chi-square test or the Kruskal–Wallis test. The P values less than 0.05 were considered statistically significant. Results We identified 198 premenopausal women who had been diagnosed with breast cancer. Thirty-eight (19%) women had TRN cancer, 67 (34%) had HER2+ cancer, and 93 (47%) had ER+ cancer. Mammographic density and cancer visibility were similar between all immunophenotypes of cancers. TRN cancers were more frequently associated with a mass (33/33 [100%]) than were HER2+ (35/64 [55%]) and ER+ cancers (42/87 [48%]) (P < 0.0001), and were less frequently associated with calcifications (5/33 [15%]) than were HER2+ (43/64 [67%]) and ER+ (53/87 [61%]) cancers (P < 0.0001). Associated ductal carcinoma in situ was reported in 18% (7/38), 57% (38/67), and 48% (52/93) of TRN, HER2+, and ER+ patients, respectively (P = 0.0003). Conclusion The mammographic features of TRN breast cancer suggest more rapid carcinogenesis leading directly to invasive cancer, that may require adjunct imaging tools for early diagnosis.     

Inhibition of autophagy and induction of breast cancer cell death by mefloquine,an antimalarial agent

Autophagy has been recognized as a potential target for cancer therapy. The antimalarial drug chloroquine (CQ) is able to inhibit autophagy and therefore is being considered for cancer therapeutics. However, the relatively low potency of CQ prompted us to investigate whether other lysosomotropic agents might be more effective, and thus potentially more useful. We therefore compared the cytotoxic efficacy of CQ, the quinoline analog mefloquine (MQ), and the fluoroquinolones ciprofloxacin and levofloxacin in several human breast cancer cell lines. We found that MQ was the most potent compound tested; it inhibited autophagy, triggered endoplasmic reticulum stress, and caused cell death in T47D and MDA-MB-231. Altogether, our study demonstrates superior potency of MQ over CQ and the ability of MQ to produce anticancer effects in both hormone receptor positive and negative breast cancer cell lines, suggesting its usefulness in treating various types of cancer.     

The evolving role of aromatase inhibitors in breast cancer

 Anti-aromatase agents inhibit the cytochrome P-450 component of the aromatase enzyme complex responsible for the final step of estrogen biosynthesis in peripheral tissues. These drugs can be classified into first-generation (e.g., aminoglutethimide), second-generation (e.g., formestane and fadrazole), and third-generation (e.g., anastrozole, letrozole, and exemestane) agents. Anti-aromatase agents can also be divided into type I and type II inhibitors. Type I inhibitors have a steroidal structure similar to androgens and inactivate the enzyme irreversibly by blocking the substrate-binding site, and are therefore known as aromatase inactivators. Type II inhibitors are nonsteroidal and their action is reversible. This article reviews the recent evidence regarding the role of third-generation aromatase inhibitors in the management of breast cancer. Relevant PubMed listed articles and presentations at recent international symposia were reviewed. There is a growing body of evidence supporting the role of third-generation aromatase inhibitors (anastrozole, letrozole, and exemestane) as first-line and second-line therapy for estrogen receptor (ER)- and/or progesterone receptor (PgR)-positive advanced breast cancer in postmenopausal women, and as a neoadjuvant therapy in postmenopausal women with hormone receptor-positive invasive breast cancer unsuitable for breast-conserving surgery. Furthermore, the preliminary results of the ATAC (Arimidex, Tamoxifen, Alone and in Combination) study have shown that adjuvant anastrozole is superior to tamoxifen in terms of disease-free survival (DFS), adverse effects, and prevention of contralateral breast cancer in postmenopausal women with early, ER-positive breast cancer. However, longer follow-up is required to assess the long-term effects of these agents on bone mineral density, cognitive function, and overall survival prior to considering their routine use in the adjuvant setting instead of tamoxifen. The potential role of these drugs in the management of ductal carcinoma in situ (DCIS), premenopausal breast cancer, and breast cancer prevention is worth investigating. Received: January 21, 2002 / Accepted: May 7, 2002 Correspondence to:K. Mokbel     

microRNA-216a在乳腺癌中表达及对MCF-7细胞自噬的影响

目的 探讨microRNA-216a(miR-216a)在人乳腺癌组织中的表达情况,并探讨其对人乳腺癌细胞株MCF-7细胞自噬的影响和相关的分子机制。方法 应用实时定量RT-PCR方法检测30例乳腺癌组织和对应癌旁组织中miR-216a的相对表达量。进一步转染miR-216a抑制剂(AMO-216a)至MCF-7细胞中,应用qRT-PCR检测miR-216a的表达情况,采用MTT检测MCF-7细胞增殖情况,Western blot方法检测自噬相关蛋白Beclin 1的表达情况。结果 乳腺癌组织中miR-216a的表达水平明显增高;与对照组相比,转染AMO-216a后,MCF-7细胞中miR-216a水平明显降低,MCF-7细胞增殖被抑制,Beclin 1蛋白表达显著增加,差异具有统计学意义(P＜0.05)。结论 miR-216a在乳腺癌组织中表达增加,可能通过下调自噬相关蛋白Beclin 1表达来激活细胞自噬,促进乳腺癌的进展。     

Diminished feedback regulation of proteasome expression and resistance to proteasome inhibitors in breast cancer cells

Clinical trials with proteasome inhibitor Bortezomib (also named Velcade or PS-341) has shown promising results for some cancers. However, other types of cancers including breast cancer do not respond well to Bortezomib. To understand the cause of the drug resistance, we compared the regulation of proteasome expression and the sensitivity to proteasome inhibitors between human breast cancer cells and nontumorigenic mammary epithelial cells. We found that, while the endogenous expression level is much higher, the potential of feedback expression in response to proteasome inhibitors is much lower in the breast cancer cells. Furthermore, the breast cancer cells are much more resistant to proteasome inhibitors compared to the nontumorigenic mammary epithelial cells. Biochemical analysis showed that the pathway of Bortezomib-induced apoptosis is apparently defective in the breast cancer cells. Together, these results provide an explanation for the inefficacy of Bortezomib in the clinical trials for breast cancer patients. The likelihood of combination therapy with Bortezomib and other anti-cancer agents for breast cancer is also discussed.     

Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and enhances mitochondrial depolarization

Macroautophagy (autophagy), a process for lysosomal degradation of organelles and long-lived proteins, has been linked to various pathologies including cancer and to the cellular response to anticancer therapies. In the human estrogen receptor positive MCF7 breast adenocarcinoma cell line, treatment with the endocrine therapeutic tamoxifen was shown previously to induce cell cycle arrest, cell death, and autophagy. To investigate specifically the role of autophagy in tamoxifen treated breast cancer cell lines, we used a siRNA approach, targeting three different autophagy genes, Atg5, Beclin-1, and Atg7. We found that knockdown of autophagy, in combination with tamoxifen in MCF7 cells, results in decreased cell viability concomitant with increased mitochondrial-mediated apoptosis. The combination of autophagy knockdown and tamoxifen treatment similarly resulted in reduced cell viability in the breast cancer cell lines, estrogen receptor positive T-47D and tamoxifen-resistant MCF7-HER2. Together, these results indicate that autophagy has a primary pro-survival role following tamoxifen treatment, and suggest that autophagy knockdown may be useful in a combination therapy setting to sensitize breast cancer cells, including tamoxifen-resistant breast cancer cells, to tamoxifen therapy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biphasic regulation of autophagy by miR-96 in prostate cancer cells under hypoxia

Autophagy favors cell survival under hypoxia, and increasing evidence revealed that microRNAs regulate autophagy. We report here hypoxia increased the expression of miR-96 in prostate cancer cells, and miR-96 stimulated autophagy by suppressing MTOR. We found that inhibition of miR-96 abolished hypoxia-induced autophagy. Paradoxically, ectopic over-expression of miR-96 to a certain threshold, also abolished the hypoxia-induced autophagy. Further studies have shown that high levels of miR-96 inhibited autophagy through suppressing ATG7, a key autophagy-associated gene. Importantly, the miR-96 expression level threshold was determined, and the effects of miR-96 on autophagy on either side of the threshold were opposite. These data demonstrate hypoxia-induced autophagy is at least partially regulated by miR-96; miR-96 can promote or inhibit autophagy by principally inhibiting MTOR or ATG7 depending on the expression levels of miR-96. Our observation might reveal a novel regulatory mode of autophagy by microRNAs under hypoxia.     

Ultrasound (US) diagnosis of nonpalpable breast cancer

BACKGROUND: Ultrasound (US) is a standard modality to diagnose breast diseases. To elucidate the usefulness of US in the diagnosis of nonpalpable breast cancers, we reviewed the cases that were treated at our institution. METHODS: Of the 106 cancers that were operated upon at the Tsukuba University Hospital between February 2004 and March 2005, 12 cancers were nonpalpable. We reviewed their US findings, results of US-guided fine needle aspiration cytology (FNAC), core biopsy results, and histological diagnoses. RESULTS: On US, 9 tumors appeared as masses. The US observations were valuable, but a confirmatory diagnosis could not be made. US-guided FNAC was performed in 8 cases; malignancy was suspected in 6 cases. US-guided core biopsy was performed in 9 cases, and it was diagnostic in 7 cases. CONCLUSIONS: Nonpalpable breast cancers can be effectively detected by US, and the diagnosis can be confirmed by US-guided FNAC or core biopsy.     

Preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors

The cellular processes of autophagy and endoplasmic reticulum stress (ERS) appear to be interconnected, and it has been proposed that autophagy may serve to reduce ERS via removal of terminally misfolded and aggregated proteins. Conversely, there are indications that blockage of autophagy may increase ERS. Based on earlier work demonstrating that pharmacologically aggravated ERS can result in tumor cell killing, we investigated whether blockage of autophagy would enhance this effect in a therapeutically useful manner. We therefore combined chloroquine (CQ), a pharmacological inhibitor of autophagy, with other drugs known to act as ERS aggravators (ERSA), namely nelfinavir (an HIV protease inhibitor) and celecoxib (a cyclooxygenase-2 inhibitor) or its non-coxib analog 2,5-dimethyl-celecoxib (DMC), and investigated combination drug effects in a variety of breast cancer cell lines. We found that the addition of CQ resulted in synergistic enhancement of tumor cell killing by ERSA compounds, particularly in triple-negative breast cancer (TNBC) cells. This combination effect could also be confirmed in an in vivo model, where CQ boosted low-dose ERSA effects, resulting in rapid deterioration of xenografted tumors in mice. Altogether, our results indicate that combinations of an autophagy inhibitor with pharmacological ERSA (i.e. compounds that lead to ER stress aggravation) should be further explored for potential therapy of otherwise difficult-to-treat TNBC.     

Polyphenol-rich extract of Pimenta dioica berries (Allspice) kills breast cancer cells by autophagy and delays growth of triple negative breast cancer in athymic mice

Bioactive compounds from edible plants have limited efficacy in treating advanced cancers, but they have potential to increase the efficacy of chemotherapy drugs in a combined treatment. An aqueous extract of berries of Pimenta dioica (Allspice) shows promise as one such candidate for combination therapy or chemoprevention. An aqueous extract of Allspice (AAE) was tested against human breast cancer (BrCa) cells in vitro and in vivo. AAE reduced the viability and clonogenic growth of several types of BrCa cells (IC₅₀ ≤ 100 μg/ml) with limited toxicity in non-tumorigenic, quiescent cells (IC₅₀ >200 μg/ml). AAE induced cytotoxicity in BrCa was inconsistent with apoptosis, but was associated with increased levels of autophagy markers LC3B and LC3B-positive puncta. Silencing the expression of autophagy related genes (ATGs) prevented AAE-induced cell death. Further, AAE caused inhibition of Akt/mTOR signaling, and showed enhanced cytotoxicity when combined with rapamycin, a chemotherapy drug and an inhibitor of mTOR signaling. Oral administration (gavage) of AAE into athymic mice implanted with MDA-MB231 tumors inhibited tumor growth slightly but not significantly (mean decrease ~ 14%, p ≥ 0.20) if mice were gavaged post-tumor implant. Tumor growth showed a significant delay (38%) in tumor palpability and growth rate (time to reach tumor volume ≥ 1,000 mm³) when mice were pre-dosed with AAE for two weeks. Analysis of tumor tissues showed increased levels of LC3B in AAE treated tumors, indicating elevated autophagic tumor cell death in vivo in treated mice. These results demonstrate antitumor and chemo-preventive activity of AAE against BrCa and potential for adjuvant to mTOR inhibition.     

靶向沉默NOB1基因对乳腺癌细胞MCF-7生长和周期分布的影响

背景与目的:NOB1(NIN1/RPN12 binding protein 1 homolog)是2005年新克隆的一个基因,属于RNA结合蛋白,该类蛋白的功能与恶性肿瘤的发生密切相关。本研究旨在观察利用慢病毒介导的RNAi沉默NOB1基因对人乳腺癌MCF-7细胞增殖及细胞周期的影响。方法:包装表达NOB1短发夹RNA(shRNA)的慢病毒,感染MCF-7细胞,实时定量PCR和蛋白质印迹法(Western blot)验证NOB1的抑制效率;MTT和克隆形成实验检测NOB1对细胞增殖能力的影响;流式细胞术检测细胞周期的变化。结果:NOB1-shRNA慢病毒感染3 d后,可显著下调MCF-7细胞NOB1 mRNA和蛋白的表达水平,显著抑制细胞增殖和体外成瘤能力,并导致细胞周期分布紊乱,G0/G1期及G2/M期细胞增加,S期细胞减少。结论:NOB1基因通过调节细胞周期分布促进乳腺癌细胞恶性增殖,可能是乳腺癌基因治疗的分子靶点。     

Therapeutic effect of tamoxifen and energy-modulating vitamins on carbohydrate-metabolizing enzymes in breast cancer

Background Cancer cells have an abnormal energetic metabolism. One of the earliest discovered hallmarks of cancer had its roots in bioenergetics, as many tumours were found in the 1920s to exhibit a high glycolytic phenotype. An animal with cancer shows significant and progressive energy loss from the host (i.e. noncancerous) tissues, which could occur by the establishment of a systemic energy-depriving cycle involving the interaction of tumour glycolysis and host gluconeogenesis. Tamoxifen (TAM) is a nonsteroidal antioestrogen that is widely used in adjuvant therapy for all stages of breast carcinoma. To improve the therapeutic efficacy of TAM and to expand its usage in the treatment of breast cancer, it is necessary to establish an energy-enhancing programme. In order to provide sufficient energy and to prevent cancer cachexia, TAM can be supplemented with energy-modulating vitamins (EMV). In this investigation the augmentation of the efficacy of TAM by the effects of EMV supplementation on carbohydrate-metabolizing enzymes, the mitochondrial Krebs cycle and respiratory enzymes was evaluated in the mammary gland of carcinoma-bearing rats.Methods Female albino Sprague-Dawley rats were selected for the investigation. The experimental set-up included one control and four experimental groups. Mammary carcinoma was induced with 7,12- dimethyl benz(a)anthracene (25 mg), and TAM was administered orally (10 mg/kg body weight per day) along with EMV which comprised riboflavin (45 mg/kg per day), niacin (100 mg/kg per day) and coenzyme Q₁₀ (40 mg/kg per day).Results Measurements were made on tumour tissue and surrounding normal tissue in all experimental groups. Tumour tissue showed significant (P<0.05) increases in the glycolytic enzymes hexokinase, phosphoglucoisomerase and aldolase, and significant decreases in the gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-biphosphatase. In contrast, the surrounding tissue showed significant decreases in glycolytic enzymes and significant increases in gluconeogenic enzymes. The activities of the mitochondrial Krebs cycle enzymes isocitrate dehydrogenase, -ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and respiratory chain enzymes NADH dehydrogenase and cytochrome c oxidase were significantly reduced in both tumour and surrounding tissue of the mammary carcinoma-bearing rats. These biochemical disturbances were effectively counteracted by supplementation with EMV, which restored the activities of all these enzyme to their respective control levels.Conclusion Combination therapy of TAM with EMV not only alters carbohydrate metabolism but can also prevent body weight loss by enhancing the host energy metabolism.