Co-delivery with nano-quercetin enhances doxorubicin-mediated cytotoxicity against MCF-7 cells

Quercetin, the plant-derived phenolic compounds, plays a pivotal role in controlling hemostasis, by having potent antioxidant and free-radical scavenging properties. This flavonoid in combination with chemotherapeutic drugs improves the efficacy of these agents in induction of apoptosis in cancer cells. This study investigated the role of nano-quercetin (phytosome) in doxorubicin-induced apoptosis. Nanoparticles were characterized for particle size, zeta potential, scanning electron microscopy (SEM) and differential scanning calorimetric assessments. Anti-proliferative effect of formulations was evaluated by MTT assay. mRNA expression levels of target genes were measured by real time RT-PCR. The mean size of nanoparticles was 85 ± 2 nm with nearly narrow size distribution which was confirmed by SEM analysis. Our results showed that co-treatment of MCF-7 breast cancer cells with nano-quercetin and doxorubicin increased the percentage of apoptosis from 40.11 ± 7.72–58 ± 7.13 (p < 0.05). Furthermore, mRNA expression levels for downstream genes including NQO1 and MRP1 showed a marked decrease (p < 0.05). Taken together, our results suggest that phytosome technology can elevate the efficacy of chemotherapeutics by increasing the permeability of tumor cells to chemical agents. Our findings introduce a novel phytosome-dependent strategy to improve delivery of doxorubicin to the breast cancerous tissues.     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

Recycling of tumor necrosis factor-alpha receptor in MCF-7 cells

Kinetics of regulation of membrane receptors for tumor necrosis factor-alpha (TNF) in human breast adenocarcinoma MCF-7 cells was investigated. When MCF-7 cells were incubated with radioiodinated human recombinant TNF, they bound TNF specifically and accumulated it intracellularly. Preincubation of cells with native TNF up to 1 x 10(-9) M for 12 h stimulated specific binding of TNF, indicating that concentrations of membrane receptors for TNF were regulated by the ligand. Accumulation of radioactivity in cells incubated with [125I]TNF proceeded at a constant rate for up to 24 h. Kinetics of binding and internalization of TNF were similar in the presence and absence of protein synthesis for at least 1 h, suggesting that the TNF receptor was either replenished from an intracellular pool of receptors or was recycled (reutilized) during the course of TNF internalization. Data were analyzed kinetically by fitting equations of compartmental models of ligand-cell interactions with and without the term for receptor recycling. Fits were obtained only for the model with receptor recycling; absence of the term for receptor recycling resulted in physically impossible best-fit parameter values. Analysis of the best-fit parameters indicated that both internalization and recycling of the receptor were stimulated by the ligand.     

In vitro effect of extracellular AMP on MCF-7 breast cancer cells: inhibition of glycolysis and cell proliferation.

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.     

Synergistic interaction between 13-cis-retinoic acid and glucarate: activity against rat mammary tumor induction and MCF-7 cells

At high dietary levels in vivo, both 13-cis-retinoic acid and calcium glucarate inhibit the induction of rat mammary tumors by 7,12-dimethylbenz(a)anthracene. The present study shows that sub-optimal dietary levels of each, which individually have no effect on tumor induction, when combined together in the diet, significantly increases tumor latency and suppresses tumor frequency in the rat system. Weight gain of animals was similar in control and experimental groups. Furthermore, ineffective sub-optimal dosages of glucarate and 13-cis-retinoic acid interacted synergistically to inhibit the growth in vitro of the MCF-7 human breast cancer cells. By varying the concentrations of glucarate and 13-cis-retinoic acid independently, evidence was obtained that in combination glucarate may play an adjuvant role, with the retinoid as the effector. Thus, the results of this experimental animal study demonstrate for the first time the potential use in synergistic combination of 2 normal metabolites in non-toxic chemoprevention and chemotherapy.     

In situ electrochemical assessment of cytotoxicity of chlorophenols in MCF-7 and HeLa cells

An in situ electrochemical method was used to assess the cytotoxicity of chlorophenols using human breast cancer (MCF-7) and cervical carcinoma (HeLa) cells as models. On treatment with different chlorophenols, the electrochemical responses of the selected cells, resulting from the oxidation of guanine and xanthine in the cytoplasm, indicated the cell viability. In addition, the in situ in vitro electrochemical method was further compared with the traditional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Although similar cytotoxicity data were obtained from both methods, the effective concentrations of chlorophenols that inhibited 50% cell growth (EC₅₀ values) from the electrochemical method were only slightly lower than those from the MTT assay. These results indicate that the in situ in vitro electrochemical method paves a simple, rapid, strongly responsive, and label-free way to the cytotoxicity assessment of different chlorophenol pollutants.     

Total synthesis and evaluation of C25-benzyloxyepothilone C for tubulin assembly and cytotoxicity against MCF-7 breast cancer cells

The total synthesis of C25-benzyloxy epothilone C is described. A sequential Suzuki–Aldol–Yamaguchi macrolactonization strategy was utilized employing a novel derivatized C8–C12 fragment. The C25-benzyloxy analog exhibited significantly reduced biological activity in microtubule assembly and cytotoxicity assays. Molecular modeling simulations indicated that excessive steric bulk in the C25 position may reduce activity by disrupting key hydrogen bonds that are crucial for epothilone binding to β-tubulin.     

The tert-butyl dimethyl silyl group as an enhancer of drug cytotoxicity against human tumor cells

In this study, we synthesized a series of enantiomerically pure (2R,3S)-disubstituted tetrahydropyranes with diverse functional groups using known methodologies. In addition to the tert-butyl dimethyl silyl group, other common protecting groups for hydroxyl groups such as allyl, acetate, and benzoate were used to obtain appropriate derivatives. Pure compounds were evaluated in vitro against HL60 human leukemia cells and MCF7 human breast cancer cells. From the growth inhibition data a structure-activity relationship was obtained. Overall the results point to the relevant role of the tert-butyl dimethyl silyl group in the modulation of cytotoxic activity.     

The cytotoxicity and anticancer mechanisms of alterporriol L, a marine bianthraquinone, against MCF-7 human breast cancer cells

Alterporriol L, a new bianthraquinone derivative, was isolated from a marine fungus Alternaria sp. ZJ9-6B. The cytotoxic activity and anticancer mechanisms of alterporriol L towards breast cancer cells lines were detected using MTT assay, immunofluorescence, and flow cytometry. Simultaneously, the changes in morphological properties of cells were detected before and after treatment with alterporriol L by atomic force microscope (AFM) at a nanometer scale. MTT assay showed that alterporriol L could effectively inhibit the growth and proliferation, and there was a dose-dependent manner of cell death. Moreover, the alterporriol L could induce cancer cell apoptosis or necrosis. Furthermore, the reactive oxygen species, mitochondrial membrane potential, and cytosolic free calcium level were changed after treatment with alterporriol L, suggesting that alterporriol L played vital roles in breast cancer cells through destroying the mitochondrial. And all these alterations are in accord with changes of morphology detected by AFM, which suggested that the AFM is a useful tool to detect the morphological changes of the cancer cells.     

In vitro enhancement of natural and antibody-dependent lymphocyte-mediated cytotoxicity against tumor target cells by interferon.

Interferon derived from virus-infected human leukocytes or fibroblasts was found to enhance spontaneous and antibody-dependent lymphocyte cytotoxicity against human target cell lines in vitro. The greater enhancement occurred with spontaneous lymphocyte cytotoxicity. Interferon exerted its effect directly on lymphocytes; no effect on target cells was seen. The mechanism of enhancement was unclear: It did not reflect antibody production or lymphocyte proliferation. Enhancement appeared to be immunologically nonspecific, but clarification of this effect awaits further study.     

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca²+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.     

In vitro cytotoxicity of liposome-encapsulated doxorubicin: dependence on liposome composition and drug release.

We have investigated the in vitro cytotoxicity of free doxorubicin (DOX) and liposome-entrapped DOX (L-DOX) against a human ovarian carcinoma cell line (OV-1063) using a colorimetric assay. DOX was encapsulated in the inner water phase of liposomes by an ammonium sulfate-generated proton gradient. Liposomes varied in phospholipid composition but were of a similar size. It was found that the cytotoxic activity of L-DOX is substantially decreased when liposomes containing phospholipids of high phase-transition temperature (Tm) are used. The type of negatively charged headgroup did not have any significant influence on the cytotoxicity observed. Experiments using resin beads that bind free and protein-bound DOX, but do not interact with L-DOX, indicated that the cytotoxic effect is mediated by the release of drug from the liposomes into the extracellular medium; no evidence was found for direct cellular uptake of liposome-encapsulated drug. The use of the ionophore nigericin to induce the release of DOX from high-Tm liposomes increased cytotoxicity to a level comparable to free DOX, suggesting that 'remote release' techniques may substantially improve the efficiency of liposome-mediated drug delivery and allow for the full exploitation of the favorable pharmacokinetic properties of specific high-Tm formulations.     

Macrophage-mediated fungistasis in vitro: requirements for intracellular and extracellular cytotoxicity

Macrophage cytotoxicity for Cryptococcus neoformans was investigated by culturing mouse peritoneal macrophages with a thin-capsuled clone of cryptococcus under conditions permitting efficient phagocytosis. Yeast replication was quantitated by electronic particle counting after detergent lysis of macrophages, and viability was determined by quantitative plate counts. Under appropriate conditions, reproduction was completely inhibited; stasis began at 2 hr after addition of yeasts and lasted for 30 hr. During this time organisms in medium alone proliferated rapidly, doubling their number every 2.5 hr. After removal from macrophages, 60 to 100% of macrophage-inhibited cryptococci formed colonies, indicating that the cytotoxic effect was primarily fungistatic. When yeast cells were removed from macrophages, replication recommenced within 5 hr. Supernatant medium from fungistatic co-cultures was not inhibitory for fresh yeast cells. Conditions required for complete fungistasis were 1) peritoneal macrophages induced by peptone from BCG-infected mice, 2) endotoxin in nanogram per milliliter range added to serum-containing cell culture medium, 3) confluent macrophage monolayers, and 4) macrophage:cryptococci ratios of 20 to 100:1. Fungistasis occurred without phagocytosis but was more efficient when cryptococci were engulfed. For efficient fungistasis, macrophages must differentiate to and be maintained in the activated state. These results with yeast cells agree with the known requirements for macrophage effector function against neoplastic target cells.     

Monoclonal antibodies to disialogangliosides: characterization of antibody-mediated cytotoxicity against human melanoma and neuroblastoma cells in vitro

We previously reported the binding specificities of two anti-ganglioside GD2 murine monoclonal antibodies (MAbs), A1-425 and A1-267, both of which are of IgG3 isotype. A1-425 reacts specifically with ganglioside GD2, whereas A1-267 binds preferentially to GD2 but also reacts with GD3 [Tai, T., Kawashima, I., Tada, N., & Dairiki, K. (1988) J. Biochem. 103, 682-687]. In this paper, they were used for comparative analyses of antibody-mediated cytotoxicity, i.e., antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human melanoma and neuroblastoma cell lines. Melanoma cells were found to contain GD2 and/or GD3, whereas neuroblastoma cells expressed only GD2. Both antibodies induced high levels of ADCC and CDC to GD2/GD3-positive cells with human peripheral large granular lymphocytes (LGL) as effector cells and in the presence of human serum, respectively. A good correlation was obtained between the contents of disialogangliosides and the binding level of the antibodies; both melanoma and neuroblastoma cells with larger amounts of GD2/GD3 showed a higher level of antibody binding than did the cells with a smaller amount of GD2/GD3. Surprisingly, ADCC did not correlate well with the binding level of the antibodies. Thus, A1-425 showed stronger lytic activity than A1-267 in spite of the binding level of A1-425 being similar to or lower than that of A1-267 on the cell surfaces. Antigen-antibody complexes composed of GD2 and A1-425 showed higher binding levels to LGL than complexes of GD2 and A1-267. In contrast, free MAb molecules gave minimum binding to LGL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Synthesis of redox-active ferrocene pyrazole conjugates and their cytotoxicity in human mammary adenocarcinoma MCF-7 cells

The redox-active ferrocenoyl modified pyrazole ligand (3-Fc-AMP) readily coordinates to a variety of transition metal ions. Here, we describe our findings regarding the synthesis and structural characterization of iron and cobalt complexes of Fc-AMP, and the cytotoxicity profiles of these chemicals in vitro. Using the human mammary adenocarcinoma MCF-7 cell line we show that the free ligand and three metal complexes induced cytotoxicity with calculated LC₅₀s ranging from 45.8 to 73.2 μM. The toxicity of the complexes decreased as the redox potential increased. The present study demonstrates the potential chemotherapeutic promise of metal complexes of a redox-active ferrocenoyl modified pyrazole ligand on human cancer cells.