Comorbidities and the use of comedications among patients with chronic hepatitis C in Korea: A nationwide cross-sectional study.

BACKGROUND/AIMS: Chronic hepatitis C (CHC) is the second leading cause of liver-related mortality and is more prevalent in the elderly population in Korea. Decisions to initiate treatment and selection of proper antiviral agents may be challenging among elderly patients due to relevant comorbidities, comedications, and drug-drug interaction (DDI). It may be helpful to understand the current demographic status and comorbidities of CHC patients in the country. METHODS: Patients aged ≥ 18 years and diagnosed with CHC (KCD-7 code B18.2) were extracted from the Korean Health Insurance Review & Assessment Service database in 2018. Data on comorbidities and comedications were assessed and potential DDIs were analyzed. RESULTS: A total of 50,476 patients with CHC, with a mean age of 60.3 years and 46.7% male patients were identified. The proportion of patients with cirrhosis, hepatocellular carcinoma, and liver transplantation was 6.0%, 4.1%, and 0.3%, respectively and 37.2% of patients were more than 65 years of age. The three most common comorbidities were diseases of the digestive system (83.7%), respiratory system (58.2%), and musculoskeletal system and connective tissue (57.6%). The three most common comedications were analgesics (91.6%), gastrointestinal agents (85%), and antibacterials (80.3%). Lipid-lowering agents and anticonvulsants were prescribed in 28.5% and 14.8% of patients. Rate of potential DDI for contraindication was 2.2%, 13.1%, and 15.6% with sofosbuvir/velpatasvir, ledipasvir/sofosbuvir, and glecaprevir/pibrentasvir. CONCLUSION: With the increasing age of patients with CHC, comorbidity, comedication, and potential DDI should be considered when choosing antivirals in Korea. Sofosbuvir-based regimens showed favorable DDI profiles among Korean patients.

Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Ganglioside GM3 suppresses lipopolysaccharide-induced inflammatory responses in rAW 264.7 macrophage cells through NF-κB, AP-1, and MAPKs signaling.

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase-2 (COX-2) protein and mRNA levels in lipopolysaccharide (LPS)-activated RAW 264.7 cells in a dose-dependent manner. Moreover, GM3 inhibited the expression and release of pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS-induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein (AP)-1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen-activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS-activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages by suppression of NF-κB, AP-1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.

Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells.

The disialic acid-containing glycosphingolipid GD3 recruited membrane transglutaminase 2 (TG2) as a signaling molecule for erythroid differentiation in human chronic myelogenous leukemia (CML) K562 cells. The α1-adrenergic receptor (α1-AR)/TG2-mediated signaling pathway regulated GD3 functions, including gene expression and production, to differentiate CML K562 cells into erythroid lineage cells. Epinephrine, an AR agonist, increased membrane recruitment as well as GTP-photoaffinity of TG2, inducing GD3 synthase gene expression. Epinephrine activated PI3K/Akt signaling and GTPase downstream of TG2 activated Akt. The coupling of TG2 and GD3 production was specifically suppressed by prazosin (α1-AR antagonist), but not by propranolol (ß-AR antagonist) or rauwolscine (α2-AR antagonist), indicating α1-AR specificity. Small interfering RNA (siRNA) experiment results indicated that the α1-AR/TG2-mediated signaling pathway activated PKCs α and δ to induce GD3 synthase gene expression. Transcription factors CREB, AP-1, and NF-κB regulated GD3 synthase gene expression during α1-AR-induced differentiation in CML K562 cells. In addition, GD3 synthase gene expression was upregulated in TG2-transfected cells via α1-AR with expression of erythroid lineage markers and benzidine-positive staining. α1-AR/TG2 signaling pathway-directed GD3 production is a crucial step in erythroid differentiation of K562 cells and GD3 interacts with α1-AR/TG2, inducing GD3/α1-AR/TG2-mediated erythroid differentiation. These results suggest that GD3, which acts as a membrane mediator of erythroid differentiation in CML cells, provides a therapeutic avenue for leukemia treatment.

Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells.

The disialoganglioside GD3 has been considered to be involved in tumor progression or suppression in various tumor cells. However, the significance of the biological functions of GD3 in breast cancer cells is still controversial. This prompted us to study the possible relationship(s) between GD3 expression and the metastatic potential of a breast cancer MDA-MB231 cells as an estrogen receptor negative (ER-) type. The human GD3 synthase cDNA was transfected into MDA-MB231 cells, and G-418 bulk selection was used to select cells stably overexpressing the GD3 synthase. In vitro invasion potentials of the GD3 synthase over-expressing cells (pc3-GD3s) were significantly suppressed when compared with control cells. Expression of intercellular adhesion molecule-1 (ICAM-1; CD54) was down-regulated in the pc3-GD3s cells and the decrease in ICAM-I expression is directly related to the decrease in invasiveness of the pc3-GD3s cells. Another type of ER negative SK-BR3 cells exhibited the similar level of ICAM-1 expression as MDA-MB231 cells, while the ER positive MCF-7 cells (ER+) showed the increased expression level of ICAM-1. Then, we investigated signaling pathways known to control ICAM-1 expression. No difference was observed in the phosphorylation of ERK and p38 between the pc3-GD3s and control cells (pc3), but the activation of AKT was inhibited in pc3-GD3s, and not in the control (pc3). In addition, the composition of total gangliosides was changed between control (pc3) and pc3-GD3s cells, as confirmed by HPTLC. The pc3-GD3s cells had an accumulation of the GD2 instead of the GD3. RT-PCR results showed that not only GD3 synthase, but also GM2/GD2 synthase (ß4-GalNc T) expression was increased in pc3-GD3s cells. Overexpression of GD3 synthase suppresses the invasive potential of human breast cancer MDA-MB-231 cells through down-regulation of ICAM-1 and the crucial pathway to allow the apoptotic effect has been attributed to accumulation of the GD2 ganglioside. ER has been linked to the ICAM-1 expression with GD3 to GD2 conversion in human breast cancer cells. This is the first finding of the endogenous sialyltransferase functions in tumor cells.

Ascofuranone inhibits lipopolysaccharide-induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1ß in RAW 264.7 macrophages.

The natural fungal compound ascofuranone (5-chloro-3-[(2E,6E)-7-[(2S)-5,5-dimethyl-4-oxo-tetrahydrofuran-2-yl]-3-methyl-octa-2,6-dienyl]-2,4-dihydroxy-6-methyl-benzaldehyde, MW 420.93) (AF) isolated from Ascochyta viciae has been known to promote cell cycle arrest and inhibit invasion of tumor cells. We have previously studied a structurally similar compound ascochlorin (ASC; MW 404.93) with regard to its anti-inflammatory activity in LPS- stimulated RAW 264.7 macrophages. In order to examine the relationship between the anti-inflammatory activities and the molecular differences between AF and ASC, the activity of AF is herein studied, because ASC has a unique trimethyl oxocyclohexyl structure, while AF has a unique dimethyl-oxo-tetrahydrofuran structure. AF dose-dependently inhibited the production of NO and iNOS and the COX-2 mRNA and protein levels in RAW 264.7 cells. In addition, AF suppressed mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and IL-1ß, as assessed by RT-PCR. AF (30-50 µg/ml) treatment clearly inhibited the nuclear translocation of NF-κB, AP-1 (p-c-Jun) from the cytosolic space. Phosphorylation of IκB, which functions to maintain the activity of NF-κB, was decreased by AF treatment. Moreover, AF suppressed the binding of NF-κB (p65). Inhibition of IkBa phosphorylation and degradation inhibits nuclear translocation of p65. Immunofluorescence confocal microscopy analysis also revealed that translocation of NF-κB and AP-1 (p-c-Jun) was decreased upon AF treatment. AF specifically decreased the expression level of p-ERK, but not the expression level of p-p38 or p-JNK. Given these results, we suggest that AF suppresses the inflammatory response by targeting p-ERK. This indicates that AF is a negative regulator of LPS-stimulated nuclear translocation of NF-κB and AP-1 (p-c-Jun) in RAW 264.7 macrophages, and specifically it targets p-ERK. Therefore, AF and ASC exert their effects in different ways, most probably because their structural differences allow for specific recognition and inhibition of their target MAPKs. Our results further suggest that AF could be a natural bioactive compound useful for treating inflammation-mediated pathological diseases.

Monosialyl Ganglioside GM3 Decreases Apolipoprotein B-100 Secretion in Liver Cells.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells.

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: ß1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.

Ganglioside GM3 participates in the TGF-ß1-induced epithelial-mesenchymal transition of human lens epithelial cells.

TGF-ß (transforming growth factor-ß)-induced EMT (epithelial-mesenchymal transition) induces the proliferation and migration of the HLE (human lens epithelial) cells. Ganglioside GM3, simple sialic-acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumour progression. However, the relationship between ganglioside GM3 and TGF-ß-induced EMT in the HLE B-3 cells is poorly understood. In the present study we demonstrated that ganglioside GM3 was involved in TGF-ß1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-ß1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of the GM3 synthase gene was regulated by Sp1 (specificity protein 1) in HLE B-3 cells upon TGF-ß1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP [d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol] and GM3 synthase shRNA (short hairpin RNA) resulted significantly in the suppression of cell migration and EMT-related signalling in HLE B-3 cells stimulated by TGF-ß. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-ß1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFßRs (TGF-ß receptors) in TGF-ß1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-ß1 regulates EMT by potential interaction with TGFßRs.

Citreorosein inhibits production of proinflammatory cytokines by blocking mitogen activated protein kinases, nuclear factor-κB and activator protein-1 activation in mouse bone marrow-derived mast cells.

Citreorosein (CIT), an anthraquinone component of Polygoni cuspidati (P. cuspidati) radix, suppressed gene expression of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the molecular mechanisms underlying CIT inhibition of the pro-inflammatory cytokine production, its effects on the activation of both nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were assessed. CIT attenuated phosphorylation of the MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase and c-Jun NH(2)-terminal kinase (JNK). Furthermore, CIT strongly inhibited DNA binding activity of NF-κB through the inhibition of phosphorylation and degradation of inhibitor of kappaB (IκB) as well as activator protein-1 (AP)-1 through the reduction of phosphorylation of c-Jun. These results demonstrate that CIT inhibits proinflammatory cytokines production through the inhibition of both MAPKs and AKT-mediated IκB kinase (IKK) phosphorylation and subsequent inhibition of transcription factor NF-κB activation, thereby attenuating the production of proinflammatory cytokines.

Triple inhibitory activity of Cliona celata against TNF-α-induced matrix metalloproteinase-9 production via downregulated NF-κB and AP-1, enzyme activity, and migration potential.

Extracellular matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), is known to be involved in vascular smooth muscle cell (SMC)'s aberrant proliferation and movement in atherosclerotic lesions. During screening of the MMP-9-inhibitory compounds from marine animal resources, we have found that the ethyl acetate extract from Cliona celata (ECC) effectively inhibits the SMC-derived MMP-9 enzyme activity and gene expression. In addition, the ECC effectively repressed the migration potential of the tumor necrosis factor-α (TNF-α)-stimulated human aortic smooth muscle cell (HASMC). As assessed by Western blot analysis, the produced MMP-9 protein levels in the TNF-α-induced HASMC were significantly decreased by the concomitant treatment of ECC at the 50- to 300-µg/mL concentration ranges. In addition, in the RT-PCR experiment, the expressed MMP-9 mRNA levels in the TNF-α-induced HASMC were seemingly decreased by ECC treatment at the same concentration ranges (50-300 µg/mL). For the action mechanism(s) of ECC to the phenotype changes in HASMC, we have further evaluated the ECC's pharmacological activities on the signal molecules which are importantly linked in the MMP-9 expression and cell migration potential of HASMC. We have found that extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation as a target point is suppressed by the ECC treatment in the TNF-α-treated HASMC. Using electrophoretic mobility shift assay, the nuclear extracts purified from ECC-treated HASMCs were shown to decrease the binding potentials on the labeled nuclear factor-kappaB (NF-κB) and activator protein 1 probes. NF-κB p65 and phosphorylated c-Jun contents were also decreased in the purified nuclear extracts from the ECC-treated HASMC, as confirmed by Western blot analysis. Finally, it was shown that the ECC-treated HASMCs were less migrated when compared to the TNF-α-treated cells, as confirmed by HASMC migration assays using the 8-µm pore transwell membranes. From these results, it was proposed that ECC has a potentially applicable anti-atherosclerotic activity.

Ethylacetate fraction from Korean seaside starfish, Asterias amurensis, has an inhibitory effect on MMP-9 activity and expression and on migration behavior of TNF-α induced human aortic smooth muscle cells.

Atherosclerosis is accompanied by the proliferation of human aortic smooth muscle cells (HASMC) and their movement into the intima. Many reports have indicated the involvement of gelatinases (MMP-9 and MMP-2) in this pathogenesis. The ethylacetate fraction from starfish, Asterias amurensis (EFA), harvested from the Korean seaside has an inhibitory effect on MMP-9 and MMP-2 activities, as well as on the expression of MMP-9 in TNF-α induced HASMC in a dose-dependent manner. Also, EFA inhibits the migration of TNF-α induced HASMC in transwells containing gelatin coated plugs. EFA was not cytotoxic to HASMC over the range 0-1mg/ml. By Western-blot analysis, it was revealed that the phosphorylation of extracellular signal regulated kinase (ERK) in TNF-α induced cells was inhibited and nuclear factor kappa B (NF-κB) p65 levels in nuclear extracts were decreased by EFA treatment. In addition, ERK inhibitor (U0126) treated cells exhibited decreased MMP-9 activity in the zymographic assay. From these results, it was found that the gelatinolytic activity was regulated (1) by enzymatic inhibition of both MMP-9 and MMP-2, as well as (2) by the decreased production of MMP-9 via ERK pathways in EFA treated HASMCs. Taken together, it has been shown that EFA has a putative anti-atherosclerotic effect.

GD3 synthase overexpression enhances proliferation and migration of MDA-MB-231 breast cancer cells.

The disialoganglioside G(D3) is an oncofetal marker of a variety of human tumors including melanoma and neuroblastoma, playing a key role in tumor progression. G(D3) and 9-O-acetyl-G(D3) are overexpressed in approximately 50% of invasive ductal breast carcinoma, but no relationship has been established between disialoganglioside expression and breast cancer progression. In order to determine the effect of G(D3) expression on breast cancer development, we analyzed the biosynthesis of gangliosides in several breast epithelial cell lines including MDA-MB-231, MCF-7, BT-20, T47-D, and MCF10A, by immunocytochemistry, flow cytometry, and real-time PCR. Our results show that, in comparison to tumors, cultured breast cancer cells express a limited pattern of gangliosides. Disialogangliosides were not detected in any cell line and G(M3) was only observed at the cell surface of MDA-MB-231 cells. To evaluate the influence of G(D3) in breast cancer cell behavior, we established and characterized MDA-MB-231 cells overexpressing G(D3) synthase. We show that G(D3) synthase expressing cells accumulate G(D3), G(D2), and G(T3) at the cell surface. Moreover, G(D3) synthase overexpression bypasses the need of serum for cell growth and increases cell migration. This suggests that G(D3) synthase overexpression may contribute to increasing the malignant properties of breast cancer cells.