Understanding the metabolic basis of drug resistance: Therapeutic induction of the Warburg effect kills cancer cells

Previously, we identified a form of epithelial-stromal metabolic coupling, in which cancer cells induce aerobic glycolysis in adjacent stromal fibroblasts, via oxidative stress, driving autophagy and mitophagy. In turn, these cancer-associated fibroblasts provide recycled nutrients to epithelial cancer cells, “fueling” oxidative mitochondrial metabolism and anabolic growth. An additional consequence is that these glycolytic fibroblasts protect cancer cells against apoptosis, by providing a steady nutrient stream to mitochondria in cancer cells. Here, we investigated whether these interactions might be the basis of tamoxifen-resistance in ER(+) breast cancer cells. We show that MCF7 cells alone are Tamoxifen-sensitive, but become resistant when co-cultured with hTERT-immortalized human fibroblasts. Next, we searched for a drug combination (Tamoxifen + Dasatinib) that could over-come fibroblast-induced Tamoxifen-resistance. Importantly, we show that this drug combination acutely induces the Warburg effect (aerobic glycolysis) in MCF7 cancer cells, abruptly cutting off their ability to use their fuel supply, effectively killing these cancer cells. Thus, we believe that the Warburg effect in tumor cells is not the “root cause” of cancer, but rather it may provide the necessary clues to preventing chemoresistance in cancer cells. Finally, we observed that this drug combination (Tamoxifen + Dasatinib) also had a generalized anti-oxidant effect, on both co-cultured fibroblasts and cancer cells alike, potentially reducing tumor-stroma co-evolution. Our results are consistent with the idea that chemo-resistance may be both a metabolic and stromal phenomenon that can be overcome by targeting mitochondrial function in epithelial cancer cells. Thus, simultaneously targeting both (1) the tumor stroma and (2) the epithelial cancer cells, with combination therapies, may be the most successful approach to anti-cancer therapy. This general strategy of combination therapy for overcoming drug resistance could be applicable to many different types of cancer.Key words: drug resistance, tamoxifen, dasatinib, tumor stroma, microenvironment, Warburg effect, aerobic glycolysis, mitochondrial oxidative phosphorylation, glucose uptake, oxidative stress, reactive oxygen species (ROS), cancer-associated fibroblasts     

Glycolysis as a metabolic marker in orthotopic breast cancer,monitored by in vivo (13)C MRS

Enhanced glycolysis represents a striking feature of cancers and can therefore serve to indicate a malignant transformation. We have developed a noninvasive, quantitative method to characterize tumor glycolysis by monitoring (13)C-labeled glucose and lactate with magnetic resonance spectroscopy. This method was applied in MCF7 human breast cancer implanted in the mammary gland of female CD1-NU mice and was further employed to assess tumor response to hormonal manipulation with the antiestrogen tamoxifen. Analysis of the kinetic data based on a unique physiological-metabolic model yielded the rate parameters of glycolysis, glucose perfusion, and lactate clearance in the tumor, as well as glucose pharmacokinetics in the plasma. Treatment with tamoxifen induced a twofold reduction in the rate of glycolysis and of lactate clearance but did not affect the other parameters. This metabolic monitoring can thus serve to evaluate the efficacy of new selective estrogen receptor modulators and may be further extended to improve diagnosis and prognosis of breast cancer.     

NADPH: Quinone oxidoreductase 1 (NQO1) mediated anti-cancer effects of plumbagin in endocrine resistant MCF7 breast cancer cells

BackgroundPLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion.Hypothesis/PurposeSince PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells.Study DesignThis study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells).MethodsThe roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR.Results1. In this study, NQO1 was upregulated in endocrine-resistant cells.2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity.3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor.4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced.5. The anti-cancer effects of PLB increased when NQO1 was upregulated.ConclusionThe effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1’s activity.     

A role of SIPL1/SHARPIN in promoting resistance to hormone therapy in breast cancer

SIPL1 inhibits PTEN function and stimulates NF-κB signaling; both processes contribute to resistance to hormone therapy in estrogen receptor positive breast cancer (ER + BC). However, whether SIPL1 promotes tamoxifen resistance in BC remains unclear. We report here that SIPL1 enhances tamoxifen resistance in ER + BC. Overexpression of SIPL1 in MCF7 and TD47 cells conferred tamoxifen resistance. In MCF7 cell-derived tamoxifen resistant (TAM-R) cells, SIPL1 expression was upregulated and knockdown of SIPL1 in TAM-R cells re-sensitized the cells to tamoxifen. Furthermore, xenograft tumors produced by MCF7 SIPL1 cells but not by MCF7 empty vector cells resisted tamoxifen treatment. Collectively, we demonstrated a role of SIPL1 in promoting tamoxifen resistance in BC. Increases in AKT activation and NF-κB signaling were detected in both MCF7 SIPL1 and TAM-R cells; using specific inhibitors and unique SIPL1 mutants to inhibit either pathway significantly reduced tamoxifen resistance. A SIPL1 mutant defective in activating both pathways was incapable of conferring resistance to tamoxifen, showing that both pathways contributed to SIPL1-derived resistance to tamoxifen in ER + BCs. Using the Curtis dataset of breast cancer (n = 1980) within the cBioPortal database, we examined a correlation of SIPL1 expression with ER + BC and resistance to hormone therapy. SIPL1 upregulation strongly associates with reductions in overall survival in BC patients, particularly in patients with hormone naïve ER + BCs. Taken together, we provide data suggesting that SIPL1 contributes to promote resistance to tamoxifen in BC cells through both AKT and NF-κB actions.     

Disruption of the ER-α36-EGFR/HER2 Positive Regulatory Loops Restores Tamoxifen Sensitivity in Tamoxifen Resistance Breast Cancer Cells

Tamoxifen provided a successful treatment for ER-positive breast cancer for many years. However, most breast tumors develop tamoxifen resistance and are eventually refractory to tamoxifen therapy. The molecular mechanisms underlying development of tamoxifen resistance have not been well established. Recently, we reported that breast cancer cells with high levels of ER-α36, a variant of ER-α, were resistant to tamoxifen and knockdown of ER-α36 expression in tamoxifen resistant cells with the shRNA method restored tamoxifen sensitivity, indicating that gained ER-α36 expression is one of the underlying mechanisms of tamoxifen resistance. Here, we found that tamoxifen induced expression of ER-α36-EGFR/HER2 positive regulatory loops and tamoxifen resistant MCF7 cells (MCF7/TAM) expressed enhanced levels of the loops. Disruption of the ER-α36-EGFR/HER2 positive regulatory loops with the dual tyrosine kinase inhibitor Lapatinib or ER-α36 down-regulator Broussoflavonol B in tamoxifen resistant MCF7 cells restored tamoxifen sensitivity. In addition, we also found both Lapatinib and Broussoflavonol B increased the growth inhibitory activity of tamoxifen in tumorsphere cells derived from MCF7/TAM cells. Our results thus demonstrated that elevated expression of the ER-α36-EGFR/HER2 loops is one of the mechanisms by which ER-positive breast cancer cells escape tamoxifen therapy. Our results thus provided a rational to develop novel therapeutic approaches for tamoxifen resistant patients by targeting the ER-α36-EGFR/HER2 loops.     

MTA1 is a novel regulator of autophagy that induces tamoxifen resistance in breast cancer cells

Tamoxifen is commonly used to treat patients with ESR/ER-positive breast cancer, but its therapeutic benefit is limited by the development of resistance. Recently, alterations in macroautophagy/autophagy function were demonstrated to be a potential mechanism for tamoxifen resistance. Although MTA1 (metastasis-associated 1) has been implicated in breast tumorigenesis and metastasis, its role in endocrine resistance has not been studied. Here, we report that the level of MTA1 expression was upregulated in the tamoxifen resistant breast cancer cell lines MCF7/TAMR and T47D/TR, and knockdown of MTA1 sensitized the cells to 4-hydroxytamoxifen (4OHT). Moreover, knockdown of MTA1 significantly decreased the enhanced autophagy flux in the tamoxifen resistant cell lines. To confirm the role of MTA1 in the development of tamoxifen resistance, we established a cell line, MCF7/MTA1, which stably expressed MTA1. Compared with parental MCF7, MCF7/MTA1 cells were more resistant to 4OHT-induced growth inhibition in vitro and in vivo, and showed increased autophagy flux and higher numbers of autophagosomes. Knockdown of ATG7 or cotreatment with hydroxychloroquine, an autophagy inhibitor, restored sensitivity to 4OHT in both the MCF7/MTA1 and tamoxifen resistant cells. In addition, AMP-activated protein kinase (AMPK) was activated, probably because of an increased AMP:ATP ratio and decreased expression of mitochondrial electron transport complex components. Finally, publicly available breast cancer patient datasets indicate that MTA1 levels correlate with poor prognosis and development of recurrence in patients with breast cancer treated with tamoxifen. Overall, our findings demonstrated that MTA1 induces AMPK activation and subsequent autophagy that could contribute to tamoxifen resistance in breast cancer.     

Metabolic activation triggered by cAMP in MCF-7 cells generates lethal vulnerability to combined oxamate/etomoxir

BackgroundAltered energy metabolism is a biochemical fingerprint of cancer cells, widely recognized as one of the “hallmarks of cancer”. Cancer cells show highly increased rates of glucose uptake and glycolysis, after which the resulting pyruvate is converted to lactate. The maintenance of this metabolic asset is warranted by lactate dehydrogenase A (LDH-A) and for this reason the development of novel LDH-targeted anticancer therapeutics is underway. However, possible interference in cancer cell metabolism could also arise from cAMP signaling pathway, which could be activated by either oncogenic induction or exogenously, as a result of microenvironment-derived stimuli, increasing cellular cAMP levels. This study aimed at evaluating the impact of activated cAMP signaling pathway on the efficacy of an LDH-targeted anticancer approach.MethodsWe exogenously activated cAMP signaling in MCF-7 human breast cancer cells and explored the metabolic interplay between LDH-A and cAMP pathway.ResultsIn cAMP-activated cells, we evidenced changes in energy metabolism which reduced their response to LDH inhibition. Interestingly, these experiments also highlighted a potential vulnerability state of treated cells.ConclusionscAMP-induced metabolic changes made MCF-7 cells a preferential target of a drug combination treatment which should not affect normal cell viability.General significancecAMP is a well-recognized second messenger of the pro-inflammatory cascade. The obtained results are relevant in consideration of the crucial role played by inflammation in normal breast cell transformation and in cancer progression. Furthermore, they corroborate the idea of exploiting the metabolic changes observed in cancer cells to obtain a therapeutic advantage.     

LIPOSOMAL DAUNORUBICIN OVERCOMES DRUG RESISTANCE IN HUMAN BREAST,OVARIAN AND LUNG CARCINOMA CELLS

ABSTRACT

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC₅₀ 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC₅₀ 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC₅₀ 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC₅₀ of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC₅₀ 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC₅₀ 237 nM) and H69VP small-cell lung carcinoma cells (IC₅₀ 27 nM). Empty liposomes did not affect the IC₅₀ for free DR in the three resistant cell lines, nor did empty liposomes affect the IC₅₀ for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.     

Prolylcarboxypeptidase regulates proliferation, autophagy, and resistance to 4-hydroxytamoxifen-induced cytotoxicity in estrogen receptor-positive breast cancer cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.Key words: caveolin-1, oxidative stress, pseudohypoxia, lactate shuttle, MCT1, MCT4, metabolic coupling, tumor stroma, predictive biomarker, SLC16A1, SLC16A3, monocarboxylic acid transporter     

Understanding the metabolic basis of drug resistance

Previously, we identified a form of epithelial-stromal metabolic coupling, in which cancer cells induce aerobic glycolysis in adjacent stromal fibroblasts, via oxidative stress, driving autophagy and mitophagy. In turn, these cancer-associated fibroblasts provide recycled nutrients to epithelial cancer cells, "fueling" oxidative mitochondrial metabolism and anabolic growth. An additional consequence is that these glycolytic fibroblasts protect cancer cells against apoptosis, by providing a steady nutrient stream of to mitochondria in cancer cells. Here, we investigated whether these interactions might be the basis of tamoxifen-resistance in ER(+) breast cancer cells. We show that MCF7 cells alone are Tamoxifen-sensitive, but become resistant when co-cultured with hTERT-immortalized human fibroblasts. Next, we searched for a drug combination (Tamoxifen + Dasatinib) that could over-come fibroblast-induced Tamoxifen-resistance. Importantly, we show that this drug combination acutely induces the Warburg effect (aerobic glycolysis) in MCF7 cancer cells, abruptly cutting off their ability to use their fuel supply, effectively killing these cancer cells. Thus, we believe that the Warburg effect in tumor cells is not the "root cause" of cancer, but rather it may provide the necessary clues to preventing chemo-resistance in cancer cells. Finally, we observed that this drug combination (Tamoxifen + Dasatinib) also had a generalized anti-oxidant effect, on both co-cultured fibroblasts and cancer cells alike, potentially reducing tumor-stroma co-evolution. Our results are consistent with the idea that chemo-resistance may be both a metabolic and stromal phenomenon that can be overcome by targeting mitochondrial function in epithelial cancer cells. Thus, simultaneously targeting both (1) the tumor stroma and (2) the epithelial cancer cells, with combination therapies, may be the most successful approach to anti-cancer therapy. This general strategy of combination therapy for overcoming drug resistance could be applicable to many different types of cancer.     

靶向AXL克服肺腺癌EGFR-TKIs获得性耐药

目的:探索AXL在肺腺癌细胞(Lung adenocarcinoma cell, LAC)EGFR-TKIs获得性耐药中的作用,为肺癌临床治疗和新型药物的研发提供实验依据。方法:构建EGFR-TKIs获得性耐药的肺腺癌模型并通过CCK-8法检测耐药株对肺腺癌靶向治疗药物吉非替尼(Gefitinib)、厄洛替尼(Erlotinib)和奥希替尼(Osimertinib)的敏感性。基于基因组学分析筛选出潜在的克服耐药的靶点AXL,通过Western blot和qRT-PCR技术检测AXL的表达情况,并同时检测上皮-间质转化(Epithelial-mesenchymal transition,EMT)分子标志物。R428是AXL的小分子抑制剂,通过CCK-8法、Transwell以及划痕实验等探究靶向AXL对肺腺癌亲本及耐药株增殖和迁移能力的影响。结果:AXL在构建的耐药株中显著高表达,其蛋白表达水平上调15-20倍(P0.001),m RNA水平上调2-5倍(P0.01);EGFR-TKIs耐药株发生上皮间质转化(EMT);靶向AXL选择性抑制耐药株的增殖能力并且恢复了耐药株对EGFR-TKIs的敏感性(P0.001);靶向AXL显著抑制耐药株增强的迁移能力,与亲本株相比最高抑制率可达80%左右(P0.001)。结论:用遗传学和药理学手段靶向AXL可以显著逆转肺腺癌对EGFR-TKIs耐药,逆转耐药株所增强的迁移等肿瘤生物学特征,对克服EGFR-TKIs获得性耐药有着重要的临床治疗价值以及转化医学前景。     

Synthesis and growth inhibition activity of fluorinated derivatives of tamoxifen

The design and synthesis of 11 fluorinated derivatives of tamoxifen are described. Growth inhibition values (GI₅₀) on human HT-29, M21, MCF7, and MDA-MB-231 tumor cells are also reported. In general, the GI₅₀ values are similar or slightly higher than tamoxifen with the most active compound on MCF7 cell line having a GI₅₀ = 3.6 μM. Surprisingly, as opposed to tamoxifen, both geometrical isomers behave similarly. We hypothesize that this behavior is due to in vitro isomerization of the compounds.     

Evidence for a stromal-epithelial “lactate shuttle” in human tumors

Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the “reverse Warburg effect,” which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, “energy transfer” or “metabolic-coupling” between the tumor stroma and epithelial cancer cells “fuels” tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.     

An <Emphasis Type="Italic">in vitro</Emphasis> model for the development of acquired tamoxifen resistance

The development of resistance to tamoxifen (Tam) remains a challenging clinical problem for ER+ breast cancer patients. To understand the mechanisms underlying of resistance, previous studies have driven the acquisition of Tam resistance by exposing cells to varying concentration of drug for varying lengths of time. However, a detailed protocol for the establishment of Tam-resistant cells remains to be clarified. In the present study, we aimed to determine and compare the effect of different in vitro protocols on the degree of resistance to 4-hydroxytamoxifen (4-OH Tam) for MCF7 cells. For this purpose, MCF7-Tam resistance (MCF7-TamR) cells were developed by treated with different concentrations (100, 200, 400, 600, 800 and 1000 nM) of 4-OH Tam over 3 months. The relative resistance was measured by WST-1 analysis. Studies characterizing of the 4-OH Tam resistance of MCF7-TamR cells were performed by 17 β-oestradiol (E2) and Annexin V/PI analysis. In addition, the expression levels of ABCC1, ABCG2 and ABCG1 were detected by RT-PCR, any changes in morphological of each resistance group were observed at the end of each month and compared with parental MCF7 cells. Consequently, exposure time and concentration can affect the degree of resistance to 4-OH Tam; thus, dose and treatment duration should be chosen according to the desired degree of resistance. This work presents a novel procedure for the generation of MCF7-TamR cells, thus enabling the identification and characterization of MCF7-TamR cells.     

Identification of eight genes that are potentially involved in tamoxifen sensitivity in breast cancer cells

Although the antiestrogen agent tamoxifen has long been used to treat women with hormone receptor positive invasive breast carcinoma, the mechanisms of its action and acquired resistance to tamoxifen during treatment are largely unknown. A number of studies have revealed that over-activation of some signaling pathways can cause tamoxifen resistance; however, very little information is available regarding the genes whose loss-of-function alternation contribute to tamoxifen resistance. Here we used a forward genetic approach in vitro to generate tamoxifen resistant cells from the tamoxifen sensitive breast cancer cell line ZR-75-1, and further identified the disrupted gene in different tamoxifen resistant clones. Retinol binding protein 7, DNA polymerase-transactivated protein 3, γ-glutamyltransferase-like activity 1,slit-robo RhoGTPase-activating protein, tetraspan NET-4, HSPC194, amiloride-sensitive epithelial sodium channel gene,and Notch2, were the eight mutated genes identified in different tamoxifen resistant clones, suggesting their requirement for tamoxifen sensitivity in ZR-75-1 cells. Since the functions of these genes are not related to each other, it suggests that multiple pathways can influence tamoxifen sensitivity in breast cancer ceils.     

Oxidative stress and glyoxalase I activity mediate dicarbonyl toxicity in MCF-7 mamma carcinoma cells and a tamoxifen resistant derivative

BackgroundAcquired tamoxifen resistance is a significant problem in estrogen receptor positive breast cancer. In a cellular model, tamoxifen resistance was associated with increased sensitivity towards toxic dicarbonyls and reduced free sulfhydryl group content. We here analyzed the role of oxidative stress and glyoxalase I activity on dicarbonyl resistance and the significance of glyoxalase I expression for survival.MethodsReactive oxygen species were determined by 2,7-dihydrochlorofluorescein diacetate. Inhibitors for NADPH-oxidase (diphenyleneiodonium), p38 MAPK (SB203580) and ERK1/2 (UO126) were applied to investigate interactions of these signaling molecules. N-acetyl cysteine was used to evaluate the effect of oxidative stress on cell viability, which was assessed by the resazurin assay. Gene expression was analyzed by real time qRT-PCR. Glyoxalase activity was inhibited by the specific inhibitor CS-0683 and siRNA. The relevance of glyoxalase 1 mRNA abundance on survival of breast cancer patients was evaluated by the KM-plotter web interface.Resultsα-Oxo-aldehydes caused an immediate increase in reactive oxygen species where the tamoxifen resistant cell line (TamR) responded at lower concentrations than the MCF-7 parental cell line. Inhibitor studies placed ROS production by NADPH-oxidase downstream of p38 MAPK. The antioxidant N-acetyl cysteine (NAC) increased survival, whereas glyoxalase (GLO1) inhibition increased dicarbonyl toxicity. GLO1 mRNA abundance was correlated with unfavorable prognosis of breast cancer patients.ConclusionsDicarbonyl toxicity was mediated by oxidative stress and GLO1 activity determines aldehyde toxicity in tamoxifen resistant cells.General SignificanceGlyoxalases might be predictive biomarkers for tamoxifen resistance and a putative target for the treatment of tamoxifen resistant breast cancer patients.     

Intracellular distribution of tamoxifen in resistant human breast adenocarcinoma cells using tamoxifen–eosin association

A tamoxifen-resistant cell line (MCF7TAM) was established from tamoxifen-sensitive MCF-7 human breast cancer cells expressing estrogen receptors. Though the resistant cell line grows in the presence of tamoxifen, estrogen receptors continue to be expressed at similar levels as in the parental cell line. However, estrogen receptors appeared to be altered in the resistant cell line since important discrepancies are observed between results obtained with ligand binding assays and immunoenzymatic assays, tending to show modifications of estrogen receptor ligand binding capacity. The intracellular distribution of tamoxifen in sensitive and resistant cell lines was investigated using fluorescence of eosin–tamoxifen ionic association. Fluorescence emission spectra of eosin, tamoxifen and eosin–tamoxifen complex (ex = 480 nm) were analyzed and the maximal fluorescence intensity found for the complex (em = 540 nm) was four times higher than that of eosin alone, while tamoxifen alone did not emit any fluorescence in this spectral range. In MCF-7 cells, tamoxifen was found to be mainly located surrounding the nucleus, although nuclear fluorescence intensity was significantly lower. No highly fluorescent granules were observed in the resistant cell lines as opposed to sensitive cells. Improvement of this fluorescence microscopy methodology could appear of interest, taking into account the complexity of tamoxifen resistance molecular pathways.