Adriamycin In MCF-7 Research Articles

IMB5036 overcomes resistance to multiple chemotherapeutic drugs in human cancer cells through pyroptosis by targeting the KH-type splicing regulatory protein

AimsIMB5036 is a pyridazinone compound with antiproliferative and antitumour activity against hepatoma and pancreatic cancer. In this study, we attempted to identify the target protein of IMB5036 and test its potential for overcoming multidrug resistance and inducing pyroptosis. Materials and methodsWe examined the effects of IMB5036 on cancer cells by in vitro assays, a molecular docking model and in vivo tumour models. We performed pull-down experiments using biotinylated IMB5036 and identified the binding proteins. Gene knockdown were used to investigate the oncogenic role of KH-type splicing regulatory protein (KSRP). Western blot was used to detect for mechanism-associated molecules. Key findingsIMB5036 could overcome resistance to multiple chemotherapeutic drugs at the cellular level and in vivo. Furthermore, IMB5036 was not a P-glycoprotein (P-gp) substrate and downregulated the expression of P-gp. We identified KSRP as a binding protein of IMB5036. The knockdown of KSRP inhibited the proliferation of MCF7 and MCF7/adriamycin (MCF7/ADR) cells. In addition, IMB5036 induced pyroptosis in both MCF7 and MCF7/ADR cells via KSRP. SignificanceWe found IMB5036 binds to KSRP and overcomes multidrug resistance via gasdermin E (GSDME)-dependent pyroptosis.

Helioscopianoids A–Q, bioactive jatrophane diterpenoid esters from Euphorbia helioscopia

The EtOH extracts of the whole plants of Euphorbia helioscopia afforded 17 new jatrophane diterpenoid esters, helioscopianoids A–Q (117), along with eight known compounds (18–25). Their structures were elucidated by extensive spectroscopic methods and Mo2(OAc)4-induced ECD analysis, and the structures of compounds 1, 2, and 7 were confirmed by X-ray crystallography. Compounds 1–17 were evaluated for inhibitory effects on P-glycoprotein (P-gp) in an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR) and neuroprotective effects against serum deprivation-induced and rotenone-induced PC12 cell damage. Compounds 8 and 16 increased the accumulation of ADM in MCF-7/ADR cells by approximately 3-fold at a concentration of 20 μmol/L. Compound 8 could attenuate rotenone-induced PC12 cell damage, and compounds 2, 8, and 12 showed neuroprotective activities against serum deprivation-induced PC12 cell damage.

Linc00518 Contributes to Multidrug Resistance Through Regulating the MiR-199a/MRP1 Axis in Breast Cancer

Background/Aims: Long non-coding RNAs (LncRNAs) have been validated to be pivotal mediators in multidrug resistance (MDR) of various cancers. This study aims to explore the roles and molecular mechanisms of linc00518 implicated in chemoresistance in breast cancer. Methods: Expressions of linc00518, miR-199a and MRP1 were evaluated by RT-qPCR or western blot. IC₅₀ values of adriamycin (ADR), vincristine (VCR) and paclitaxel (PTX) were determined by XTT assays and cell apoptosis was assessed by flow cytometry. Luciferase reporter and RIP assays were employed to detect the interaction of linc00518, miR-199a and MRP-1. Results: linc00518 expression increased nearly 2 fold and MRP1 level elevated about 2.5 fold in breast cancer tissues as compared to that in adjacent normal tissues. Also, almost 2 fold upregulation of linc00518 and MRP-1 expressions was observed in MCF-7 cells than in MCF-10A cells. Additionally, linc00518 level was almost 2.5 fold higher and MRP1 level was about 2 fold increased in ADR-resistant MCF-7 cells (MCF-7/ADR) than in parental cell line MCF-7. Linc00518 knockdown enhanced chemosensitivity to ADR, VCR and PTX, and boosted ADR-, VCR- and PTX-induced apoptosis in MCF-7/ADR cells. miR-199a inhibitor conferred chemoresistance to ADR, VCR and PTX in MCF-7/ADR cells, and suppressing miR-199a reversed multi-drug susceptibility induced by linc00518 knockdown. Furthermore, linc00518 could act as a molecular sponge of miR-199a to repress MRP1 expression. MRP1 depletion increased the sensitivity of MCF-7/ADR cells to ADR, VCR and PTX, and this effect was attenuated following miR-199a inhibition or linc00518 overexpression. Also, linc00518 silencing increased ADR-mediated anti-tumor effect in vivo. Conclusions: linc00518 downregulation reduced MDR by regulating miR-199a/MRP1 axis in breast cancer.

Galectin-1 knockdown improves drug sensitivity of breast cancer by reducing P-glycoprotein expression through inhibiting the Raf-1/AP-1 signaling pathway.

Galectin-1 (Gal-1), a member of the galectin family of carbohydrate binding proteins, plays a pivotal role in various cellular processes of tumorigenesis. The regulatory effect of Gal-1 on multidrug resistance (MDR) breast cancer cells is still unclear. qRT-PCR and western blot showed that Gal-1 and MDR gene 1 (MDR1) were both highly expressed in breast tumor tissues and cell lines. MTT assay and flow cytometry revealed that Gal-1 knockdown improved sensitivity to paclitaxel (PTX) and adriamycin (ADR) in MCF-7/PTX and MCF-7/ADR cells via inhibition of cell viability and promotion of cell apoptosis, while MDR1 overexpression weakened the sensitivity to PTX and ADR induced by Gal-1 knockdown. Furthermore, the negative effects of Gal-1 knockdown on sensitivity to PTX and ADR in MCF-7/PTX and MCF-7/ADR cells were revealed to be mediated via the suppression of Raf-1/AP-1 pathway. In conclusion, Gal-1 knockdown dramatically improved drug sensitivity of breast cancer by reducing P-glycoprotein (P-gp) expression via inhibiting the Raf-1/AP-1 pathway, providing a novel therapeutic target to overcome MDR in breast cancer.

Dioscin strengthens the efficiency of adriamycin in MCF-7 and MCF-7/ADR cells through autophagy induction: More than just down-regulation of MDR1.

The purpose of present study was to investigate the effect of dioscin on activity of adriamycin (ADR) in ADR-sensitive (MCF-7) and ADR-resistant (MCF-7/ADR) human breast cancer cells and to clarify the molecular mechanisms involved. Antiproliferation effect of ADR was enhanced by dioscin in MCF-7 and MCF-7/ADR cells. Dioscin significantly inhibited MDR1 mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activity in MCF-7/ADR cells. Additionally, inhibitor κB-α (IκB-α) degradation was inhibited by dioscin. Moreover, dioscin induced the formation of vacuoles in the cytoplasm and protein level of LC3-II in MCF-7 and MCF-7/ADR cells. Autophagy inhibitor 3-MA abolished the effect of dioscin on ADR cytotoxicity. Dioscin inhibited phosphorylation of PI3K and Akt, resulting in upregulation of LC3-II expression. In conclusion, dioscin increased ADR chemosensitivity by down-regulating MDR1 expression through NF-κB signaling inhibition in MCF-7/ADR cells. Autophagy was induced by dioscin to ameliorate the cytotoxicity of ADR via inhibition of the PI3K/AKT pathways in MCF-7 and MCF-7/ADR cells. These findings provide evidence in support of further investigation into the clinical application of dioscin as a chemotherapy adjuvant.

Neo-Clerodane diterpenoids from Scutellaria barbata mediated inhibition of P-glycoprotein in MCF-7/ADR cells

Ten new (1-10) and seventeen known (11-27) neo-clerodane diterpenoids substituted with nicotinoyloxyl were isolated from the plant Scutellaria barbata and their structures were established by extensive spectroscopic analysis. Chemoreversal effects of these neo-clerodane diterpenoids on multidrug resistance were evaluated in breast cancer multidrug-resistant MCF-7/ADR cells that overexpress P-glycoprotein. Four compounds (11, 14, 16, and 18) exhibited better chemoreversal abilities than the classical P-gp inhibitor verapamil and the most potent compound 11 reduced IC50 value of adriamycin in MCF-7/ADR cells from 58.8 μM to 1.3 μM. Mechanistic investigations showed that compound 11 reversed multidrug resistance through suppressing the activity of P-gp and restraining the expression of P-glycoprotein. In the present study, the structure–activity relationships of neo-clerodane diterpenoids were also discussed.

Inhibitory Effects of Highly Oxygenated Lanostane Derivatives from the Fungus Ganoderma lucidum on P-Glycoprotein and α-Glucosidase.

Twelve new highly oxygenated lanostane triterpenoids and nine known ganoderic acids were isolated from the fruiting body of Ganoderma lucidum. The new compounds were lanostane nortriterpenoids with 27 carbons (1-5 and 8), lanostane nor-triterpenoids with 25 carbons (6 and 7), and lanostane triterpenoids (9-12) based on multiple spectroscopic data analysis, including HRESIMS, 1D-NMR, 2D-NMR, and CD. Compounds 1-5 were identified as rare nor-lanostanoids that contain a 17β-pentatomic lactone ring. Compound 13, possessing a lactone ring, had been isolated previously. The P-glycoprotein (P-gp) inhibitory effects of compounds 1-21 were evaluated at a concentration of 20 μM using an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR). Compounds 1, 5, 18, and 20 and verapamil increased the accumulation of ADM in MCF-7/ADR cells approximately 3-fold when compared with the negative control. These data support the significant P-glycoprotein inhibitory activities of compounds 1, 5, 18, and 20. In silico docking analysis suggested these compounds had similar P-gp recognition mechanisms compared with those of verapamil (a classical inhibitor). Furthermore, in an in vitro bioassay, compounds 2, 4, 5, 6, and 18 showed moderate inhibitory effects against α-glucosidase compared with those of the positive control acarbose.

Microbial transformation of diosgenin by Cunninghamella blakesleana AS 3.970 and potential inhibitory effects on P-glycoprotein of its metabolites

Microbial transformation of diosgenin ((25R)-spirost-5-en-3β-ol) using Cunninghamella blakesleana AS 3.970, afforded eleven polyhydroxylated derivatives. Compounds 4 and 6 could increase the accumulation of adriamycin in MCF-7/ADR cells.

Recombinant human interleukin 24 reverses Adriamycin resistance in a human breast cancer cell line

BackgroundThe major cause of multidrug resistance is over-expression of membrane P-glycoprotein (P-gp). We investigated the effect of recombinant human interleukin 24 (rhIL-24) on the Adriamycin (ADM)-resistant human breast cancer cell line MCF-7/ADM. MethodsThe cytotoxicity of rhIL-24 and ADM was determined by 3-[4,5-dimethylthiazol-2-yl], 5-diphenyl tetrazolium bromide (MTT) assays. The expression of P-gp was assessed by confocal microscopy and Western blot analysis. ResultsThe IC50 values for rhIL-24 in MCF-7/wild-type and MCF-7/ADM cells were 0.17 and 14.6μM, respectively. The IC50 value of Adriamycin in MCF-7/ADM cells decreased in a dose-dependent manner when rhIL-24 was used. The resistance modulating factor (RMF) was directly proportional to the dose of rhIL24. ADM accumulation increased while P-gp expression decreased at a low dose (4μM) of rhIL24 in MCF-7/ADM cells. The expression of P-gp was decreased at 4μM in confocal microscopy and western blot analysis. ConclusionsrhIL-24 circumvented the drug-resistance of MCF-7/ADM cells via activation of the transcription factor Stat 3. rhIl24 has potential to act as a P-gp inhibitor to reverse Adriamycin resistance in breast cancer.

PH-sensitive pullulan-based nanoparticle carrier for adriamycin to overcome drug-resistance of cancer cells

Urocanic acid was conjugated to pullulan to synthesize O-urocanyl pullulan (URPA) with degree of substitution (DS) of 8.2%. URPA nanoparticles prepared by dialysis method had spherical shapes and a mean diameter of 156.8±16.8nm. Adriamycin (ADR) was successfully loaded into URPA nanoparticles and exhibited pH-sensitive in vitro release property. MTT assay showed that ADR-loaded URPA (ADR/URPA) nanoparticles had a significant higher toxicity against drug resistant MCF-7/ADR cells than free ADR, and the reversal index reached up to 9.6. The results of flow cytometry and confocal microscopy showed that URPA nanoparticles efficiently enhanced accumulation and retention of ADR in MCF-7/ADR cells and successfully delivered ADR into cell nucleus. The reversal effect of ADR/URPA nanoparticles on the drug resistance of MCF-7/ADR cells was perhaps related with their cell entry and intracellular drug release mechanisms.

N-lauroyl chitosan surface-modified PLGA nanoparticles as carrier for adriamycin to overcome cancer drug resistance

N-lauroyl chitosan (NLCS) conjugates with different degrees of substitution (DS) of lauroyl group were synthesized and used to prepare surface modified poly(lactic-co-glycolic) acid (NLCS-PLGA) nanoparticles via hydrophobic interaction and ionic bond force. NLCS-PLGA nanoparticles had spherical shape with shell-core structure and exhibited the smallest size and narrowest size distribution when DS of lauroyl group of NLCS was 8.5%. Adriamycin (ADR), as a model antitumor drug, was loaded into NLCS-PLGA nanoaprticles and its initial burst release from PLGA nanoparticles was significantly reduced. MTT assay showed that NLCS-2-PLGA nanoaprticles evidently enhanced cytotoxicity of ADR against drug-resistant breast cancer MCF-7/ADR cells, both compared to free ADR and ADR-loaded PLGA nanoparticles. Moreover, cell-live images showed that the cellular uptake and nuclear location of ADR in MCF-7/ADR cells were significantly enhanced by loading of NLCS-2-PLGA nanoparticles. In conclusion, this novel carrier of anticancer drugs has the potential to overcome drug resistance in cancer cells.

Reversal effects of Rabdosia rubescens extract on multidrug resistance of MCF-7/Adr cells in vitro

Context: Rabdosia rubescens (Hemsl.) Hara (Lamiaceae) is widely used in traditional Chinese medicines for the treatment of antitumor, antimicrobial, anti-inflammatory and antioxidation. It is also used as a supplement in the treatment of many cancers, such as esophagus, mammary gland, liver and prostate cancers.Objective: To investigate the multidrug resistance (MDR) reversal effects and its possible mechanism of R. rubescens extracts on human breast cancer cell line MCF-7/Adr (Michigan Cancer Foundation – 7/adriamycin resistance).Materials and methods: Rabdosia rubescens were extracted by reflux extraction method with different solvent such as petroleum ether, chloroform, ethyl acetate, n-butyl alcohol and water in order and obtain petroleum ether fraction (PEF), chloroform fraction (CF), ethyl acetate fraction (EAF), n-butyl alcohol fraction (BAF) and aqueous fraction (AF). The active extract fractions of R. rubescens were screened by rhodamine123 (Rh123) accumulation assay. Cytotoxicity of the effect fraction was examined by the MTT assay; the intracellular accumulation of adriamycin and expression of P-gp were examined by flow cytometry; the gene transcription of MDR1 was determined by RT-PCR.Results: CF and EAF fractions could increase the intracellular accumulation of adriamycin in MCF-7/Adr cells, PEF, BAF and AF fractions showed little effect on the intracellular accumulation of adriamycin or Rh123. When adriamycin was used in combination with CF and EAF fractions at non-toxic concentration on MCF-7/Adr cells, CF and EAF fractions can reverse MDR of MCF-7/Adr cells, and the reverse folds were 2.16 (CF, 4 μg/mL), 4.60 (CF, 20 μg/mL), 1.87 (EAF, 4 μg/mL) and 4.02 (EAF, 20 μg/mL), respectively. After treatment with CF (4.20 μg/mL) and EAF (4.20 μg/mL) for 48 h, the MDR1 gene expression level in MCF-7/Adr cells was decreased by 40.17, 48.14, 33.86 and 42.52%, and the abundance of P-gp also decreased by 8.63, 24.53, 27.50 and 34.91% in MCF-7/Adr cells, respectively.Discussion and conclusion: These results indicate the therapeutic value of chloroform fraction (CF) and ethyl acetate fraction (EAF) from R. rubescens as potential MDR reversing agents and warranted further investigation.

Functionalized Graphene Oxide Mediated Adriamycin Delivery and miR-21 Gene Silencing to Overcome Tumor Multidrug Resistance In Vitro

Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. Co-delivery of novel MDR-reversing agents and anticancer drugs to cancer cells holds great promise for cancer treatment. MicroRNA-21 (miR-21) overexpression is associated with the development and progression of MDR in breast cancer, and it is emerging as a novel and promising MDR-reversing target. In this study, a multifunctional nanocomplex, composed of polyethylenimine (PEI)/poly(sodium 4-styrenesulfonates) (PSS)/graphene oxide (GO) and termed PPG, was prepared using the layer-by-layer assembly method to evaluate the reversal effects of PPG as a carrier for adriamycin (ADR) along with miR-21 targeted siRNA (anti-miR-21) in cancer drug resistance. ADR was firstly loaded onto the PPG surface (PPGADR) by physical mixing and anti-miR-21 was sequentially loaded onto PPGADR through electric absorption to form anti-miR-21PPGADR. Cell experiments showed that PPG significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line) and exhibited much higher cytotoxicity than free ADR, suggesting that PPG could effectively reverse ADR resistance of MCF-7/ADR. Furthermore, the enhanced therapeutic efficacy of PPG could be correlated with effective silencing of miR-21 and with increased accumulation of ADR in drug-resistant tumor cells. The endocytosis study confirmed that PPG could effectively carry drug molecules into cells via the caveolae and clathrin-mediated endocytosis pathways. These results suggest that this PPG could be a potential and efficient non-viral vector for reversing MDR, and the strategy of combining anticancer drugs with miRNA therapy to overcome MDR could be an attractive approach in cancer treatment.

The Function Role of miR-181a in Chemosensitivity to Adriamycin by Targeting Bcl-2 in Low-Invasive Breast Cancer Cells

Objectives: miR-181a is involved in immunity, metabolism, tumor suppression or carcinogenesis reported by many other studies. However, its role in the development of chemosensitivity to adriamycin in low-invasive breast cancer cells remains unclear. The aim of this study is to define the function role of miR-181a in promoting the efficacy of adriamycin-based neoadjuvant chemotherapy. Methods: Cell survival analysis was detected by Cell Counting Kit-8 assay. Apoptotic cells were quantitatively detected using FITC Annexin V apoptosis Detection Kit I. Bcl-2 protein expression was measured by western blot. Luciferase reporter vector with the putative BCL-2 3' untranslated region (3'UTR) was constructed to explore whether BCL-2 was a direct target gene of miR-181a. Real-time PCR was performed to test the expression of miR-181a and Bcl-2 in the selected breast cancer tissue samples. Results: The down-regulation of miR-181a decreased adriamycin-induced apoptosis in MCF-7 cells. Transfected with miR-181a mimic in cells resulted in the decreased expression of Bcl-2. The alteration of miR-181a expression did not significantly affect the chemosensitivity to adriamycin in MCF-7 and MCF-7/ADR cells with genetic knockout of Bcl-2. miR-181a may suppress Bcl-2 expression by forming imperfect base pairing with the 3'UTR of Bcl-2 gene such that a negative relationship between miR-181a and Bcl-2 in MCF-7 and MCF-7/ADR cells is observed. Conclusions: At least in part, the detection of miR-181a may direct the clinical medication in patients with neoadjuvant chemotherapy because of miR-181a enhanced adriamycin-induced apoptosis via targeting Bcl-2.

Graphene oxide used as a carrier for adriamycin can reverse drug resistance in breast cancer cells

This study evaluates the reversal effects of graphene oxide (GO) used as a carrier for adriamycin (ADR) in cancer drug resistance, and provides a preliminary investigation into the reversal mechanism. ADR was loaded onto the GO surface (ADR–GO) by physical mixing and drug loading content was found to be high, up to 93.6%. In vitro releases of ADR from ADR–GO were studied using a dialysis method, and they exhibited a significant pH-sensitive property. Cell experiments showed that GO significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line) and exhibited much higher cytotoxicity than free ADR, suggesting that ADR–GO could effectively reverse ADR resistance of MCF-7/ADR, with the reversal index reaching 8.35. Microscopy studies found that GO could effectively carry drug molecules into cells in both endocytosis-dependent and independent manners. In conclusion, use of GO as a carrier for chemotherapeutic agents is favorable for the treatment of drug resistant cancers.

Key Role of Nuclear Factor-κB in the Cellular Pharmacokinetics of Adriamycin in MCF-7/Adr Cells: The Potential Mechanism for Synergy with 20(S)-Ginsenoside Rh2

We have previously demonstrated that ginsenoside 20(S)-Rh2 is a potent ATP-binding cassette (ABC) B1 inhibitor and explored the cellular pharmacokinetic mechanisms for its synergistic effect on the cytotoxicity of adriamycin. The present studies were conducted to elucidate the key factors that influenced ABCB1 expression, which could further alter adriamycin cellular pharmacokinetics. Meanwhile, the influence of 20(S)-Rh2 on the above factors was revealed for explaining its synergistic effect from the view of ABCB1 expression. The results indicated that 20(S)-Rh2 inhibited adriamycin-induced ABCB1 expression in MCF-7/Adr cells. Subsequent analyses indicated that 20(S)-Rh2 markedly inhibited adriamycin-induced activation of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB pathway, NF-κB translocation to the nucleus, and NF-κB binding activity. Furthermore, 20(S)-Rh2 repressed the Adriamycin-enhanced ability of NF-κB to bind to the human multidrug resistance (MDR1) promoter, and MAPK/NF-κB inhibitors and NF-κB small interfering RNA reversed the adriamycin-induced expression of ABCB1. Moreover, the cellular pharmacokinetics of adriamycin was also significantly altered by inhibiting NF-κB. In conclusion, the MAPK/NF-κB pathway mediates adriamycin-induced ABCB1 expression and subsequently alters the cellular pharmacokinetics of adriamycin. It was speculated that 20(S)-Rh2 acted on this pathway to lower adriamycin-induced ABCB1 expression in MCF-7/Adr cells, which provided mechanism-based support to the development of 20(S)-Rh2 as a MDR reversal agent.

Cellular pharmacokinetic mechanisms of adriamycin resistance and its modulation by 20(S)‐ginsenoside Rh2 in MCF‐7/Adr cells

Intracellular pharmacokinetics of anticancer drugs in multi-drug resistance (MDR) cancer cells is hugely important in the evaluation and improvement of drug efficacy. By using adriamycin as a probe drug in MDR cancer cells, we developed a cellular pharmacokinetic-pharmacodynamic (PK-PD) model to reveal the correlation between cellular pharmacokinetic properties and drug resistance. In addition, the ability of 20(S)-ginsenoside Rh2 (20(S)-Rh2) to reverse MDR was further investigated. The cellular pharmacokinetics of adriamycin were analysed visually and quantitatively in human breast cancer cells MCF-7 and in adriamycin-resistant MCF-7 (MCF-7/Adr) cells. Mitochondria membrane potential was assayed to evaluate the apoptotic effect of adriamycin. Subsequently, a PK-PD model was developed via MATLAB. Visual and quantitative data of the dynamic subcellular distribution of adriamycin revealed that it accumulated in cells, especially nuclei, to a lesser and slower extent in MCF-7/Adr than in MCF-7 cells. 20(S)-Rh2 increased the rate and amount of adriamycin entering cellular/subcellular compartments in MCF-7/Adr cells through inhibition of P-glycoprotein (P-gp) activity, in turn augmenting adriamycin-induced apoptosis. The integrated PK-PD model mathematically revealed the pharmacokinetic mechanisms of adriamycin resistance in MCF-7/Adr cells and its reversal by 20(S)-Rh2. P-gp, which is overexpressed and functionally active at cellular/subcellular membranes, influences the cellular pharmacokinetic and pharmacological properties of adriamycin in MCF-7/Adr cells. Inhibition of P-gp activity represents a key mechanism by which 20(S)-Rh2 attenuates adriamycin resistance. Even more importantly, our findings provide a new strategy to explore the in-depth mechanisms of MDR and evaluate the efficacy of MDR modulators.

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to Adriamycin

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21 waf1/cip1 and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21 waf1/cip1, down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

Bcl-2 inhibits mitochondrial metabolism and lonidamine-induced apoptosis in adriamycin-resistant MCF7 cells.

Lonidamine (LND), a selective inhibitor of the energy metabolism of tumor cells, induces apoptosis, independently of the p53 gene, in the adriamycin(ADR)-resistant MCF7 breast-cancer cell line (MCF7 ADR). On the contrary, LND fails to activate the apoptotic program in the parental MCF7-sensitive cell line (MCF7 WT). The extent of bcl-2 expression might account for the different effect of LND on these cell lines. In fact, the MCF7 ADR line shows a low level of bcl-2 protein, whereas MCF7 WT expresses a high level of bcl-2. We therefore investigated the relationship between the amount of bcl-2 and the ability of LND to induce apoptosis, using 4 clones over-expressing bcl-2. The effect of bcl-2 on the energy metabolism was also evaluated. We demonstrated that over-expression of bcl-2 inhibited LND-induced apoptosis, while reducing 14CO2 production, oxygen uptake and ATP content, whereas aerobic lactate production was essentially unaffected. In addition, LND decreased the oxidative metabolism of the MCF7 ADR cells to a greater extent than it did in the bcl-2 transfectants.

Effects of 1,2-naphthoquinones on human tumor cell growth and lack of cross-resistance with other anticancer agents.

The sensitivity of human tumor and rat prostate tumor cells to a series of naphthoquinones, including tricyclic compounds of the beta-lapachone and dunnione families as well as 4-alkoxy-1,2-naphthoquinones, was evaluated. To better understand the mechanism of cytotoxicity of 1,2-naphthoquinones, the roles of various resistance mechanisms including P-glycoprotein, multidrug resistant associated protein, glutathione (GSH) and related enzymes, altered topoisomerase activity, and overexpression of genes that control apoptosis (bcl-2 and bc-xL) were studied. MCF7 cells were most sensitive to the naphthoquinones with IC50 values ranging from 1.1 to 10.8 microM, as compared to 2.5 to >32 microM for HT29 human colon, A549 human lung, CEM leukemia and AT3.1 rat prostate cancer cells. MCF7 ADR cells, selected for resistance to adriamycin (ADR), displayed cross-resistance to the tricyclic 1,2-naphthoquinones. Drug efflux via a P-glycoprotein mechanism was ruled out as a mechanism of resistance to 1,2-naphthoquinones, since KB-V1 cells expressing high levels of P-glycoprotein and the KB-3.1 parent line were equally sensitive to these compounds. Any resistance of the tricyclic naphthoquinones noted in ADR-resistant cells appeared to relate to the GSH redox cycle and could be circumvented by exposure to buthionine sulfoximine or by changing the structure from a tricyclic derivative to a 4-alkoxy-1,2-naphthoquinone. The 1,2-naphthoquinones were found to be cytotoxic against CEM/VM-1 and CEM/M70-B1 cells that were selected for resistance to teniposide or merbarone, respectively. In addition, cells overexpressing bcl-2 or bcl-xL proteins were as sensitive to 1,2-naphthoquinones as were control cells. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective chemotherapeutic agents.