Liposomal formulation of HIF-1α inhibitor echinomycin eliminates established metastases of triple-negative breast cancer.

Hypoxia-inducible factor 1α (HIF-1α) is recognized as a prime molecular target for metastatic cancer. However, no specific HIF-1α inhibitor has been approved for clinical use. Here, we demonstrated that in vivo efficacy of echinomycin in solid tumors with HIF-1α overexpression is formulation-dependent. Compared to previously-used Cremophor-formulated echinomycin, which was toxic and ineffective in clinical trials, liposomal-echinomycin provides significantly more inhibition of primary tumor growth and only liposome-formulated echinomycin can eliminate established triple-negative breast cancer (TNBC) metastases, which are the leading cause of death from breast cancer, as available therapies remain minimally effective at this stage. Pharmacodynamic analyses reveal liposomal-echinomycin more potently inhibits HIF-1α transcriptional activity in primary and metastasized TNBC cells in vivo, the latter of which are HIF-1α enriched. The data suggest that nanoliposomal-echinomycin can provide safe and effective therapeutic HIF-1α inhibition and could represent the most potent HIF-1α inhibitor in prospective trials for metastatic cancer.

Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets.

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.

Effects of Notch-1 down-regulation on malignant behaviors of breast cancer stem cells.

This study examined the effect of Notch-1 signaling on malignant behaviors of breast cancer cells by regulating breast cancer stem cells (BCSCs). BCSCs were enriched by using serum-free medium and knocked out of Notch-1 by using a lentiviral vector. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the Notch-1 expression levels in breast cancer cell lines and BCSCs, and flow cytometry to detect the proportion of BCSCs in BCSC spheres. The BCSC self-renewal, migration, invasion, and tumorigenicity were examined by the tumor microsphere-forming assay and transwell assay and after xenotransplantation. The results showed that the Notch-1 silencing reduced the number of BCSC spheres, the proportion of BCSCs, and the number of cells penetrating through the transwell membrane. It also decreased the size of tumors that were implanted in the nude mice. These results suggest that Notch-1 signaling is intimately linked to the behaviors of BCSCs. Blocking Notch-1 signaling can inhibit the malignant behaviors of BCSCs, which may provide a promising therapeutical approach for breast cancer.

All-potassium channel CRISPR screening reveals a lysine-specific pathway of insulin secretion.

OBJECTIVE: Genome-scale CRISPR-Cas9 knockout coupled with single-cell RNA sequencing (scRNA-seq) has been used to identify function-related genes. However, this method may knock out too many genes, leading to low efficiency in finding genes of interest. Insulin secretion is controlled by several electrophysiological events, including fluxes of KATP depolarization and K+ repolarization. It is well known that glucose stimulates insulin secretion from pancreatic ß-cells, mainly via the KATP depolarization channel, but whether other nutrients directly regulate the repolarization K+ channel to promote insulin secretion is unknown. METHODS: We used a system involving CRISPR-Cas9-mediated knockout of all 83 K+ channels and scRNA-seq in a pancreatic ß cell line to identify genes associated with insulin secretion. RESULTS: The expression levels of insulin genes were significantly increased after all-K+ channel knockout. Furthermore, Kcnb1 and Kcnh6 were the two most important repolarization K+ channels for the increase in high-glucose-dependent insulin secretion that occurred upon application of specific inhibitors of the channels. Kcnh6 currents, but not Kcnb1 currents, were reduced by one of the amino acids, lysine, in both transfected cells, primary cells and mice with ß-cell-specific deletion of Kcnh6. CONCLUSIONS: Our function-related CRISPR screen with scRNA-seq identifies Kcnh6 as a lysine-specific channel.

Polysaccharides: potential bioactive macromolecules for Alzheimer's disease.

Alzheimer's disease (AD) is one of the leading causes of death and disability. AD is a devastating disease that has caused an overwhelming burden. However, no disease-modified treatment was discovered. The approval of sodium oligomannate (GV-971) in mild-moderate AD patients has attracted great attention to investigate the role of saccharides in AD. Therefore, summarizing and explaining the role of saccharides in AD is urgent and promising. Recent studies showed that polysaccharides (PSs) potentially benefit AD in vitro and in vivo. PSs could alleviate the pathological damage and improve cognitive symptoms via (1) antagonizing the toxicity of abnormal amyloid-beta and tau proteins; (2) attenuating oxidative stress and proinflammation; (3) rebuilding neuroplasticity. PSs exhibit one-multiple pathological hits of AD. However, a thorough chemical investigation is needed for further study.

Pseudolaric acid B triggers cell apoptosis by activating AMPK/JNK/DRP1/mitochondrial fission pathway in hepatocellular carcinoma.

Pseudolaric acid B (PAB), a natural product isolated from the root bark of Pseudolarix kaempferi, has been reported to exert inhibitory effects in various cancers. However, the underlying mechanisms remain largely unclear. In the present study, we investigated the mechanism through which PAB exert its anticancer effects in hepatocellular carcinoma (HCC). PAB inhibited the viability of and induced apoptosis in Hepa1-6 cells in a dose-dependent manner. It disrupted mitochondrial membrane potential (MMP) and impaired ATP production. Furthermore, PAB induced phosphorylation of DRP1 at Ser616 and mitochondrial fission. Blocking DRP1 phosphorylation by Mdivi-1 inhibited mitochondrial fission and PAB-induced apoptosis. Moreover, c-Jun N-terminal kinase (JNK) was activated by PAB, and blocking JNK activity using SP600125 inhibited PAB-induced mitochondrial fission and cell apoptosis. Furthermore, PAB activated AMP-activated protein kinase (AMPK), and inhibiting AMPK by compound C attenuated PAB-stimulated JNK activation and blocked DRP1-dependent mitochondrial fission and apoptosis. Our in vivo data confirmed that PAB inhibited tumor growth and induced apoptosis in an HCC syngeneic mouse model by inducing the AMPK/JNK/DRP1/mitochondrial fission signaling pathway. Furthermore, a combination of PAB and sorafenib showed a synergistic effect in inhibiting tumor growth in vivo. Taken together, our findings highlight a potential therapeutic strategy for HCC.

[Effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in rats with osteoporosis and periodontitis].

PURPOSE: To investigate the effects of chitosan oligosaccharide on bone metabolism and IKK/NF-κB pathway in mice with osteoporosis and periodontitis. METHODS: Thirty rats were randomly divided into 3 groups, with 10 rats in each group. They were divided into control group, ovariectomized periodontitis group and chitosan oligosaccharide treatment group. Except for the control group, the other two groups were ovariectomized and smeared with Porphyromonas gingivalis fluid to establish the model of osteoporosis with periodontitis. Four weeks after ligation, the rats in chitosan oligosaccharide treatment group were gavaged with 200 mg/kg chitosan oligosaccharide, and the other two groups were gavaged with equal volume of normal saline once a day for 90 days. The periodontal tissues of each group were observed before administration, and the bone mineral density of rats was detected by dual energy X-ray animal bone mineral density and body composition analysis system. After 90 days of administration, the bone mineral density was detected again. After administration, blood was collected from tail vein, and the contents of serum alkaline phosphatase (ALP), bone Gla protein (BGP) and tartrate resistant acid phosphatase 5b (TRACP5b) were measured by enzyme-linked immunodeficient assay. The gingival index and periodontal attachment loss of rats in each group were obtained by visual examination and exploratory examination. The maxilla was removed, and the distance from the enamel cementum boundary to the alveolar crest was measured to obtain alveolar bone absorption value. H-E staining was used to observe the pathology of maxilla in each group. RT-PCR and Western blot were used to detect the nuclear factors in periodontal tissue of rats in each group. SPSS 22.0 software package was used for statistical analysis. RESULTS: Before administration, the gums of the control group were pink without bleeding, and the gums of the other two groups were red and swollen with slight bleeding. After administration, compared with the control group, the bone mineral density, serum ALP, BGP of ovariectomized periodontitis group decreased significantly(P＜0.05); while TRACP5b, gingival index, loss of periodontal attachment and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue increased significantly(P＜0.05). Compared with the ovariectomized periodontitis group, the bone mineral density, serum ALP, BGP were significantly increased(P＜0.05); while TRACP5b, gingival index, periodontal attachment loss and alveolar bone resorption, NF-κB and IKK mRNA and protein expression in periodontal tissue were significantly decreased (P＜0.05). In the ovariectomized periodontitis group, the periodontal tissue combined with epithelium was separated from the tooth surface, the dental pocket was obvious and deep, and the height of alveolar bone decreased. Although dental pocket could be observed in the periodontal tissue of rats treated with chitosan oligosaccharide, it was not obvious, and new bone appeared around the alveolar bone. CONCLUSIONS: Chitosan oligosaccharide can induce biochemical indexes of bone metabolism to become normal, alleviate the symptoms of periodontitis, this may be related to the inhibition of IKK/NF-κB pathway by chitosan oligosaccharide.

[Regulation mechanism of resveratrol on odontogenic differentiation of human dental pulp stem cells].

PURPOSE: To investigate whether resveratrol promotes odontogenic differentiation of human dental pulp stem cells(DPSCs) by up-regulating the expression of silent information regulator 1 (SIRT1) and activating ß-catenin signaling pathway. METHODS: Different concentrations of resveratrol(0, 10, 15, 20 and 50 µmol/L) were used to treat DPSCs for 7 days and 14 days, and cell proliferative activity was detected by CCK-8. After odontogenic differentiation induced by 15 µmol/L resveratrol for 7 days, alkaline phosphatase(ALP) staining was performed and real-time quantitative reverse transcription PCR(qRT-PCR) was used to detect the mRNA expression of Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein(DSPP) and dentin matrix protein-1(DMP-1) in DPSCs. Western blot was used to detect the expression of SIRT1 in DPSCs on a specific day (0, 3rd, 5th, 7th and 14th) after differentiation induction. Western blot was also used to detect the expression of SIRT1 and activated ß-catenin during odontogenic differentiation of DPSCs treated by 15 µmol/L resveratrol for 7 days. The experimental data was analyzed with GraphPad Prism 9 software package. RESULTS: 15 µmol/L resveratrol had no significant effect on proliferation of DPSCs on the 7th and 14th day; 15 µmol/L resveratrol promoted odontogenic differentiation of DPSCs and up-regulated mRNA expression of RUNX2, DSPP, and DMP-1 in DPSCs; the expression of SIRT1 was the highest on the 7th day during odontogenic differentiation induction. Resveratrol resulted in the increasing protein expressions of SIRT1 and activated ß-catenin when DPSCs was induced to odontogenic differentiation for 7 days. CONCLUSIONS: Resveratrol promotes odontogenic differentiation of human DPSCs by up-regulating the expression of SIRT1 protein and activating ß-catenin signaling pathway.

Identification of the major functional proteins of prokaryotic lipid droplets.

Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83-146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids.

Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells.

Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno-fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently.

Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein a-I.

The lipid droplet (LD) is a universal organelle governing the storage and turnover of neutral lipids. Mounting evidence indicates that elevated intramuscular triglyceride (IMTG) in skeletal muscle LDs is closely associated with insulin resistance and Type 2 Diabetes Mellitus (T2DM). Therefore, the identification of the skeletal muscle LD proteome will provide some clues to dissect the mechanism connecting IMTG with T2DM. In the present work, we identified 324 LD-associated proteins in mouse skeletal muscle LDs through mass spectrometry analysis. Besides lipid metabolism and membrane traffic proteins, a remarkable number of mitochondrial proteins were observed in the skeletal muscle LD proteome. Furthermore, imaging by fluorescence microscopy and transmission electronic microscopy (TEM) directly demonstrated that mitochondria closely adhere to LDs in vivo. Moreover, our results revealed for the first time that apolipoprotein A-I (apo A-I), the principal apolipoprotein of high density lipoprotein (HDL) particles, was also localized on skeletal muscle LDs. Further studies verified that apo A-I was expressed endogenously by skeletal muscle cells. In conclusion, we report the protein composition and characterization of skeletal muscle LDs and describe a novel LD-associated protein, apo A-I.

Recent Progress in Solar-Induced Direct Biomass-to-Electricity Hybrid Fuel Cell Using Microalgae as Feedstocks.

Microalgae, as potential biodiesel feedstocks, have been widely reported to accumulate oil via genetic engineering techniques, or environmental stress regulation. Recently, the utilization of fuel cell technology to convert biomass into electricity has attracted much more attention due to its high efficiency, low pollution, low noise by microalgae as feedstocks. Normally, platinum and analogous noble metals as catalysts have been already demonstrated although they still exist lots of shortcomings. This mini review presents an overview of various fuel cell technologies with phosphomolybdic acid as catalysts for sustainable energy by using microalgae. Trends from literatures demonstrate that algal-based fuel cells could efficiently generate electricity, and concurrently produce high value-added products. This critical review can provide guiding suggestions for future study of algal-based energy conversion by fuel cell techniques.

The HIF1α-PDGFD-PDGFRα axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin.

BACKGROUND: Glioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies. METHODS: Target genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence. RESULTS: HIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway. CONCLUSIONS: HIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.

[Resveratrol suppressed the expression of MIP-2 induced by lipopolysaccharide in osteoblasts via up-regulating SOCS-3].

PURPOSE: To explore whether resveratrol dependents on the production of suppressor of cytokine signaling suppressor 3 (SOCS-3) in inhibiting mRNA production of macrophage inflammatory protein-2 (MIP-2) in osteoblasts induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e). METHODS: MC3T3-E1 cells were treated with different concentrations of resveratrol (0, 5, 10 and 20 µmol/L) and 20 µmol/L resveratrol for different time( 0, 10, 30, 60, 120 and 180 min). The expression of SOCS-3 protein was detected by Western blot. MC3T3-E1 cells were transfected with mouse SOCS3 siRNA (si-SOCS-3) and control siRNA(si-control). Reverse transcription real-time PCR(real-time RT-PCR) and Western blot was used to detect the silencing efficiency of SOCS-3. Cells were stimulated by 20 µg/mL P.e-LPS for 24 h after transfection, in the absence or presence of 20 µmol/L resveratrol for 1 h , and the changes of MIP-2 mRNA were determined by real-time RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. RESULTS: Treatment of MC3T3-El cells with different concentrations of resveratrol caused a significant increase in SOCS-3 protein expression in a dose-dependent manner. During the observation time of 180 min, SOCS-3 protein expression was the highest at 20 µmol/L resveratrol-treated osteoblasts for 60 min. The silencing efficiency of SOCS-3 mRNA was 63.7%. Transfection with SOCS-3 siRNA increased MIP-2 mRNA expression in LPS-stimulated MC3T3-E1 cells and negated the inhibitory effects of resveratrol on LPS-induced MIP-2 mRNA expression(P<0.05). CONCLUSIONS: Resveratrol inhibits the expression of MIP-2 mRNA in osteoblasts induced by P.e-LPS by up-regulating the expression of SOCS-3 protein.

A role for lipid droplets in inter-membrane lipid traffic.

All cells have the capacity to accumulate neutral lipids and package them into lipid droplets. Recent proteomic analyses indicate that lipid droplets are not simple lipid storage depots, but rather complex organelles that have multiple cellular functions. One of these proposed functions is to distribute neutral lipids as well as phospholipids to various membrane-bound organelles within the cell. Here, we summarize the lipid droplet-associated membrane-trafficking proteins and review the evidence that lipid droplets interact with endoplasmic reticulum, endosomes, peroxisomes, and mitochondria. Based on this evidence, we present a model for how lipid droplets can distribute lipids to specific membrane compartments.

Extracellular ATP-induced nuclear Ca2+ transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic beta-cells.

Extracellular ATP (eATP) induces an intracellular Ca(2+) transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca(2+) release from intracellular stores in mouse pancreatic beta-cells. Using laser scanning confocal microscopy, Ca(2+) indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca(2+) release in pancreatic beta-cell nuclei. eATP induced a higher nuclear Ca(2+) transient in pancreatic beta-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca(2+) transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca(2+)-ATPase (PMCA) or the plasma membrane Na(+)/Ca(2+) exchanger (NCX) by LaCl(3) or by replacement of Na(+) with N-Methyl-Glucosamine. eATP-induced nuclear Ca(2+) transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic beta-cells. These results indicate that eATP triggers nuclear Ca(2+) transients by mobilizing a nuclear Ca(2+) store via nuclear IP3Rs.

CD8+ cytotoxic and FoxP3+ regulatory T lymphocytes serve as prognostic factors in breast cancer.

BACKGROUND: There is conflicting evidence regarding the prognostic value of cytotoxic T cell infiltration in breast cancer. The aims of this study were to detect the expression levels and localization of FoxP3 and CD8 in invasive ductal carcinoma of the breast and to investigate the correlations among FoxP3+ regulatory T cells (Tregs), CD8+ cytotoxic T lymphocytes (CTLs), clinicopathological features, and prognosis in patients with breast cancer. METHODS: Immunohistochemistry was used to detect the expression levels and localization of FoxP3 and CD8. One-sample t-test, one-way analysis of variance, and Kaplan-Meier log-rank tests were used to analyze correlations between the expression levels of CD8 and FoxP3; Kaplan-Meier Log-rank test was used to analyze clinicopathological features to explore the prognostic significance of CD8 and FoxP3 in patients with breast cancer. RESULTS: FoxP3 expression in the tumor bed was higher than that in the stroma, while CD8 was primarily expressed in the stroma. CD8 expression was associated with favorable prognostic factors. However, FoxP3 expression and an increased ratio of total FoxP3+ Tregs to CD8+ CTLs were significantly correlated with unfavorable prognostic factors. Additionally, an increased ratio was associated with molecular subtypes (ER+Her2+, ER+Her2-, ER-Her2+, and ER-Her2-) of breast cancer. Overexpression of FoxP3 and a high FoxP3+/CD8+ ratio were correlated with poor overall survival (OS) and disease-free survival (DFS). However, CD8 expression only affected OS in patients with breast cancer. CONCLUSIONS: Tumor-infiltrating lymphocytes are localized variously depending on the subtype. CD8+ CTLs were associated with a good prognosis, while FoxP3+ Tregs were associated with adverse outcomes in patients with breast cancer. CD8+ CTLs and FoxP3+ Tregs are potential predictive prognostic factors for patients with breast cancer.