Photodynamic anticancer activity evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates.

5-Aminolevulinic acid (ALA) and its derivatives, serving as the endogenous precursor of the photosensitizer (PS) protoporphyrin IX (PpIX), successfully applied in tumor imaging and photodynamic therapy (PDT). ALA and its derivatives have been used to treat actinic keratosis (AK), basal cell carcinoma (BCC), and improve the detection of superficial bladder cancer. However, the high hydrophilicity of ALA and the conversion of PpIX to heme have limited the accumulation of PpIX, hindering the efficiency and potential application of ALA-PDT. This study aims to evaluate the PDT activity of three rationally designed series of ALA-HPO prodrugs, which were based on enhancing the lipophilicity of the prodrugs and reducing the labile iron pool (LIP) through HPO iron chelators to promote PpIX accumulation. Twenty-four ALA-HPO conjugates, incorporating amide, amino acid, and ester linkages, were synthesized. Most of the conjugates, exhibited no dark-toxicity to cells, according to bioactivity evaluation. Ester conjugates 19a-g showed promoted phototoxicity when tested on tumor cell lines, and this increased phototoxicity was strongly correlated with elevated PpIX levels. Among them, conjugate 19c emerged as the most promising (HeLa, IC50 = 24.25 ± 1.43 µM; MCF-7, IC50 = 43.30 ± 1.76 µM; A375, IC50 = 28.03 ± 1.00 µM), displaying superior photodynamic anticancer activity to ALA (IC50 > 100 µM). At a concentration of 80 µM, the fluorescence intensity of PpIX induced by compound 19c in HeLa, MCF-7, and A375 cells was 18.9, 5.3, and 2.8 times higher, respectively, than that induced by ALA. In conclusion, cellular phototoxicity showed a strong correlation with intracellular PpIX fluorescence levels, indicating the potential application of ALA-HPO conjugates in ALA-PDT.

Discovery of Potent and Wild-Type-Sparing Fourth-Generation EGFR Inhibitors for Treatment of Osimertinib-Resistance NSCLC.

Osimertinib resistance is an unmet clinical need for the treatment of non-small cell lung cancer (NSCLC), and the main mechanism is tertiary C797S mutation of epidermal growth factor receptor (EGFR). To date, there is no inhibitor approved for the treatment of Osimertinib-resistant NSCLC. Herein, we reported a series of Osimertinib derivatives as fourth-generation inhibitors which were rationally designed. Top candidate D51 potently inhibited the EGFRL858R/T790M/C797S mutant with an IC50 value of 14 nM and suppressed the proliferation of H1975-TM cells with an IC50 value of 14 nM, which show over 500-fold selectivity against wild-type forms. Moreover, D51 inhibited the EGFRdel19/T790M/C797S mutant and the proliferation of the PC9-TM cell line with IC50 values of 62 and 82 nM. D51 also exhibited favorable in vivo druggability, including PK parameters, safety properties, in vivo stability, and antitumor activity.

Cysteine conjugate beta-lyase 2 (CCBL2) expression as a prognostic marker of survival in breast cancer patients.

OBJECTIVE: Cysteine conjugate beta-lyase 2 (CCBL2), also known as kynurenine aminotransferase 3 (KAT3) or glutamine transaminase L (GTL), plays an essential role in transamination and cytochrome P450. Its correlation with some other cancers has been explored, but breast cancer (BC) not yet. METHODS: The mRNA and protein expression of CCBL2 in BC cell lines and patient samples were detected by RT-qPCR and immunohistochemistry (IHC). BC patients' clinical information and RNA-Seq expression were acquired via The Cancer Genome Atlas (TCGA) database. Patients were categorized into high/low CCBL2 expression groups based on the optimal cutoff value (8.973) determined by receiver operating characteristic (ROC) curve. We investigated CCBL2 and clinicopathological characteristics' relationship using Chi-square tests, estimated diagnostic capacity using ROC curves and drew survival curves using Kaplan-Meier estimate. We compared survival differences using Cox regression and externally validated using Gene Expression Omnibus (GEO) database. We evaluated enriched signaling pathways using gene set enrichment analysis (GSEA), explored CCBL2 and relevant genes' relationship using tumor immunoassay resource (TIMER) databases and used the human protein atlas (HPA) for pan-cancer analysis and IHC. RESULTS: CCBL2 was overexpressed in normal human cell lines and tissues. CCBL2 expression was lower in BC tissues (n = 1104) than in normal tissues (n = 114), validated by GEO database. Several clinicopathologic features were related to CCBL2, especially estrogen receptor (ER), progesterone receptor (PR) and clinical stages. The low expression group exhibited poor survival. CCBL2's area under curve (AUC) analysis showed finite diagnostic capacity. Multivariate cox-regression analysis indicated CCBL2 independently predicted BC survival. GSEA showed enriched pathways: early estrogen response, MYC and so on. CCBL2 positively correlated with estrogen, progesterone and androgen receptors. CCBL2 was downregulated in most cancers and was associated with their survival, including renal and ovarian cancers. CONCLUSIONS: Low CCBL2 expression is a promising poor BC survival independent prognostic marker.

[Corrigendum] Rab22a is a novel prognostic marker for cell progression in breast cancer.

Following the publication of this article, the authors have realized that Table I contained an error: The number of patients who were alive in the Rab22a high expression group should have been written as 77 instead of 772.
A corrected version of the Table is shown on the next page (the corrected datum is highlighted in bold). The authors sincerely apologize for the error that was introduced during the preparation of this Table, and regret any inconvenience that this mistake has caused. [the original article was published in International Journal of Molecular Medicine 45: 1037-1046, 2020; DOI: 10.3892/ijmm.2020.4486].

Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1.

Aberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC. XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor.

Wntless (Wls): A Prognostic Index for Progression and Patient Survival of Breast Cancer.

BACKGROUND: Wntless (Wls) is an essential protein that is necessary for the secretion of Wnt proteins. While numerous researches have demonstrated that aberrations in Wnt/ß-catenin expression lead to tumorigenesis and progression in many cancer types, the effects of Wls in breast cancer (BC) are less studied. METHODS: The mRNA and protein expression of Wls in BC cell lines were detected by RT-qPCR and Western blot; the protein expression of patient samples was detected by immunohistochemistry (IHC). The associations between Wls expression and clinicopathological factors as well as survival time, including overall survival (OS) and disease-free survival (DFS) were analyzed. Bioinformatics analysis was used to reveal the correlation between Wls genes and associated genes or pathways. RESULTS: Wls was overexpressed in BC cell lines and tissues. The expression level of Wls was significantly correlated with tumor size, estrogen receptor (ER), progesterone receptor (PR), Ki-67, molecular classification, and follow-up status. Spearman correlation analysis showed that Wls protein expression was negatively correlated with ER and PR, which was confirmed by bioinformatics analysis in mRNA level. However, there were positive relationships with MBNG (modified Black's nuclear grade), tumor size, Ki-67, molecular classification, follow-up, and vital status. Univariate and multivariate analysis showed that Wls was an independent prognostic factor for OS and DFS in BC patients. Moreover, Wls was a significant prognostic indicator of OS and DFS in a hormone receptor-positive (HR+) subgroup. GSEA showed that estrogen and androgen response, as well as epithelial-mesenchymal transition pathways, were up-regulated in the Wls high-expression group. CONCLUSION: Overexpression of Wls is a significant marker of worse prognosis in BC and might play a crucial role in the HR+ subgroup.

Follicular dendritic cell sarcoma in the right chest wall: A case report.

RATIONALE: Follicular dendritic cell sarcoma (FDCS) is a rare malignant tumor that originates from germinal center follicular dendritic cells, and can occur at both nodal and extranodal sites. There are very few described cases of FDCS arising in the chest wall. PATIENT CONCERNS: A 44-year-old male patient presented with a history of right chest wall pain for 5 months. DIAGNOSES: Positron emission tomography/computed tomography showed a significant increase in F-fluorodeoxyglucose uptake and multiple small axillary lymph nodes without hypermetabolic lesions. Immunohistochemistry results of a core-needle biopsy indicated FDCS, which was consistent with the postoperative pathological examination. INTERVENTIONS: The patient underwent tumor resection with lymphadenectomy of level I axillary nodes. No metastasis in the lymph nodes was observed in the postoperative pathological examination. The patient did not accept chemotherapy or radiotherapy. OUTCOMES: After 18 months, the patient remains in good condition with no evidence of disease recurrence. LESSONS: This report highlights a rare case of a FDCS arising in the chest wall. Accurate clinical diagnosis and staging of this rare malignant sarcoma is essential for the developmnt of effective treatment strategies. Preoperative F-fluorodeoxyglucose positron emission tomography/computed tomography scanning combined with core-needle biopsy could provide differentiation between benign and malignant tumors, as well as lymph node involvement and metastatic status.

Rab22a is a novel prognostic marker for cell progression in breast cancer.

Breast cancer (BC) is the most common female malignant tumor worldwide. The mechanism of tumorigenesis is still unclear. Ras­related proteins in brain (Rab)22a belongs to the Ras superfamily, which may act as an oncogene and participate in carcinogenesis. The present study aims to identify whether Rab22a could be a novel biomarker of prognosis and determine the effects of Rab22a on BC cell progression. A total 258 BC and 56 para­tumor or non­tumor formalin fixed paraffin embedded tissues were stained through immunohistochemistry. The association between Rab22a expression and clinicopathological features, as well as overall survival status were analyzed. The expression level of Rab22a in breast cell lines were detected using reverse transcription­quantitative PCR and western blotting. SK­BR­3 cells were infected with Rab22a short hairpin RNA lenti­virus and the ability of cell proliferation, migration and invasion were measured. Gene Set Enrichment Analysis (GSEA) was employed to analyze the pathways involved in the Rab22a mRNA high level group. Rab22a was found to be overexpressed in BC tissues and upregulated in BC cells. High expression of Rab22a was related to a poor prognosis of patients with BC. Knockdown of Rab22a decreased the proliferation, migration and invasion ability of BC cells. GSEA indicated that certain pathways, including mammalian target of rapamycin complex 1 and protein secretion were upregulated, while pathways, such as hypoxia and KRas were downregulated in the Rab22a high level group. Rab22a is of prognostic value for BC and necessary for BC cell proliferation.

Does Choice of Reconstruction Type Affect Survival in Patients With Metastatic Breast Cancer?

BACKGROUND: To compare the breast cancer-specific survival (BCSS) and overall survival (OS) between patients who underwent implant or tissue reconstruction after mastectomy with distant metastatic breast cancer (MBC). MATERIALS AND METHODS: Using the Surveillance, Epidemiology, and End Results database, we enrolled 371 female MBC cases diagnosed during the years 2004-2014. Patients were subdivided into implant (176) and tissue groups (195) for survival comparison between the two groups. The end points were BCSS and OS. Comparisons of the distribution of clinicopathologic characteristics were evaluated by chi-square test and Fisher exact test. Survival outcomes were compared by Kaplan-Meier model and multivariate Cox regression model for known clinicopathologic variables in both the entire population and in the reconstruction cohorts. RESULTS: In the entire cohort, patients with implant exhibited distinctly better BCSS (log rank, P = 0.002) and OS (log rank, P = 0.001) than patients with tissue reconstruction. Multivariate Cox regression model revealed that patients, who received prosthetic implants, were married, and progesterone receptor-positive group showed better survival rates in BCSS and OS. In addition, after stratification of the implant group and tissue groups according to clinicopathologic variables, the survival rate of patients in the implant group was higher than that in the tissue reconstruction group under the influence of factors, such as married, estrogen receptor-negative, nonradiotherapy, and chemotherapy. CONCLUSIONS: Our study provides further survival evidence supporting the practice of mastectomy with prosthetic implant reconstruction in patients with MBC under certain conditions.

A new prognostic factor of breast cancer: High carboxyl ester lipase expression related to poor survival.

OBJECTIVE: The enzyme carboxyl ester lipase (CEL), known as bile salt-dependent lipase (BSDL) or bile salt-stimulated lipase (BSSL), is mainly expressed in pancreatic acinar cells and lactating mammary glands. To investigate the link between CEL expression of breast cancer (BC) tissues and the survival of BC patients by analyzing The Cancer Genome Atlas Breast Carcinoma (TCGA-BRCA) level 3 data. METHODS: The clinical information and RNA-sequencing (RNA-Seq) expression data were downloaded from TCGA. Patients were divided into a high CEL expression group and a low CEL expression group using the optimal cutoff value (5.611) identified from the ROC curve. Chi-square test and Fisher exact test were used to find the correlation between the expression of CEL and clinicopathologic features. To assess the diagnostic capability, the receiver operating characteristic (ROC) curve of CEL was drawn. The survival differences between high and low CEL expression groups were compared by Cox regression analysis. Log-rank test was applied to the calculation of p values and the comparison of the Kaplan-Meier curves. Furthermore, Gene Expression Omnibus (GEO) datasets were used for external data validation. RESULTS: Analysis of 1104 cases of tumor data showed that CEL was over-expressed in breast cancer. There were relationships between high CEL expression and clinicopathologic features. The high CEL expression group had a lower survival. By analyzing the area under the ROC curve (AUC) of CEL, it was found to have a limited diagnostic capability. CEL expression may be an independent prognostic factor for breast cancer survival through the multivariate analysis. The validation in GEO datasets also showed that CEL expression was higher in breast tumor tissues than in normal breast tissues. High CEL expression was associated with the poor overall survival of breast cancer. CONCLUSIONS: High CEL expression may be an independent prognostic factor for the poor survival of breast cancer.

Comprehensive multigene mutation spectra of breast cancer patients from Northeast China obtained using the Ion Torrent sequencing platform.

The objectives of the present study were to obtain the multigene mutation spectra of female breast cancer patients in Northeast China, to explore the correlation between mutations and clinicopathological characteristics, and to identify genetic mutations that correlate with the prognosis and survival of breast cancer patients. An Ion Torrent sequencing platform was used to detect mutations, including 31 known gene mutations associated with breast cancer, in 621 specimens from 286 breast cancer patients. A total of 286 patients were enrolled in this study. Eleven harmful/pathogenic gene mutations were found in 54.2% (155/286) of the patients, and 179 somatic nonsynonymous mutations were detected. Approximately 5.6% (16/286) of the patients carried two or more gene mutations. Among the 11 pathogenic gene mutations, those in PIK3CA were the most common and were detected in 65.4% (117/179) of the patients; TP53 gene mutations were the second most common and were detected in 20.7% (37/179) of the patients. Additional mutations were found in AKT (14/179; 7.8%) and PTEN (4/179; 2.2%), and mutations in the remaining 7 genes were each detected in approximately 0.6% (1/179) of the patients. Excluding 6 cases of breast ductal carcinoma in situ, the remaining 280 breast cancer cases were divided into four groups by molecular subtype, and the mutation frequencies of the 11 breast cancer­associated genes differed among the four groups. Furthermore, these 280 breast cancer cases were divided into two clinically relevant therapeutic groups: the HR+/HER2­ and triple­negative groups. The triple­negative group had a high frequency of TP53 mutations (21.8%) and a low frequency of PIK3CA mutations (21.8%), whereas the HR+/HER2­ group harbored TP53 mutations at a low frequency (10.1%) and PIK3CA mutations at a high frequency (50.0%). Cancerous, paracancerous, and normal tissues were collected from 72 patients and subjected to next­generation sequencing. The types and frequencies of somatic nonsynonymous mutations differed among the three studied tissue types, reflecting the genetic heterogeneity of different tissues from the same individual. In addition, tissues from 70 patients (excluding 2 patients with ductal carcinoma in situ) were divided into four groups according to molecular subtype, and the gene mutation frequencies in cancerous, paracancerous, and normal tissues differed among the four groups. After normalization, gene mutations were detected at a higher rate in cancerous tissues than in paracancerous and normal tissues in all groups, except for the HER2­positive group (which had a small sample size). In addition, Cox multivariate analyses of clinicopathological data, gene sequencing results, and 5­year survival rates of the 286 patients showed that gene mutations in the PTEN­PI3K/AKT signaling pathway were independently associated with a poor prognosis (P<0.05). In conclusion, mutations in the PTEN­PI3K/AKT signaling pathway may be valuable in the prediction of the prognosis and survival of breast cancer patients.

MicroRNA-206 inhibits metastasis of triple-negative breast cancer by targeting transmembrane 4 L6 family member 1.

Purpose: Breast cancer (BC) is a common malignancy in women, but the survival rate for BC is not very encouraging. Especially for triple-negative breast cancer (TNBC), a kind of breast cancer that does not have any of the receptors that are commonly found in BC. We investigated the impact of microRNA-206 (miR-206) on transmembrane 4 L6 family member 1 (TM4SF1) in TNBC for therapeutic purpose. Patients and methods: Twenty BC tissues from diagnosed BC patients were analyzed via real-time PCR and Western blotting for expression of TM4SF1 and miR-206. The expression of TM4SF1 was studied in relationship with miR-206 in MDA-MB-231 cells. The biological impact of TM4SF1 and miR-206 on MDA-MB-231 cells and BALB/c nude mice model was studied using proliferation, transwell migration, and invasion assays both in vitro and in vivo. Results: The expression of TM4SF1 in BC tissues was significantly higher than that in adjacent normal breast tissues. In contrast, miR-206 showed a decreased expression level in BC tissues, especially for subtype basal like. Overexpression of miR-206 in MDA-MB-231 cells by transfecting miR-206 resulted in downregulation of TM4SF1. In contrast, knockdown miR-206 expression reversed miR-206-mediated phenotype in MDA-MB-231 cells. Expression level of TM4SF1 in MDA-MB-231 cells was associated with cell migration and invasion capabilities in vitro. Breast tumor burden was correlated with the expression level of TM4SF1 in vivo. Conclusion: Taken together, our results showed the involvement of TM4SF1 in TNBC migration and invasion. miR-206 negatively regulated gene expression of TM4SF1. These findings indicate that miR-206 could be used as a potential therapeutic agent for TNBC.

A large and aggressive fibromatosis in the axilla: a rare case report and review of the literature.

Aggressive fibromatosis (AF) is a rare benign tumor, which occurs in the deep part of bone and muscle fibrous tissue. Clinical and pathological features can be challenging for definitive diagnosis. Here, we report a rare case of a large AF in the axilla. Interestingly, 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography showed significant increase in standard uptake value. Surgical resection yielded a spindle cell tumor likely of fibromatosis origin which was positive for ß-catenin expression.

Downregulation of the long non-coding RNA ZFAS1 is associated with cell proliferation, migration and invasion in breast cancer.

Long non-coding RNAs (lncRNAs) are non­coding RNAs that are >200 nucleotides in length. Recent studies have identified a number of lncRNAs with critical roles in various biological processes including tumorigenesis. Zinc finger antisense 1 (ZFAS1) is a lncRNA that has recently been reported to be involved in the progression of several human cancers. However, the biological function of ZFAS1 in breast cancer remains to be elucidated. In order to determine the effect of ZFAS1 in breast cancer cells, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to measure ZFAS1 expression in cells from breast cancer cell lines. In addition, gain­of­function experiments were performed in vitro to investigate the biological role of ZFAS1. The results revealed that ZFAS1 expression was significantly downregulated in breast cancer cell lines when compared with the levels in controls. In vitro experiments also demonstrated that ZFAS1 overexpression significantly suppressed cell proliferation by causing cell cycle arrest and inducing apoptosis in breast cancer cells. Further functional assays indicated that ZFAS1 overexpression inhibited cell migration and invasion by regulating epithelial­mesenchymal transition. These findings indicated that the lncRNA ZFAS1 may be a tumor suppressor in breast cancer, and thus, may serve as a potential therapeutic target for patients with breast cancer.

Design, synthesis and biological evaluation of phthalimide-alkylamine derivatives as balanced multifunctional cholinesterase and monoamine oxidase-B inhibitors for the treatment of Alzheimer's disease.

A series of novel phthalimide-alkylamine derivatives were synthesized and evaluated as multi-functions inhibitors for the treatment of Alzheimer's disease (AD). The results showed that compound TM-9 could be regarded as a balanced multi-targets active molecule. It exhibited potent and balanced inhibitory activities against ChE and MAO-B (huAChE, huBuChE, and huMAO-B with IC50 values of 1.2µM, 3.8µM and 2.6 µM, respectively) with low selectivity. Both kinetic analysis of AChE inhibition and molecular modeling study suggested that TM-9 binds simultaneously to the catalytic active site and peripheral anionic site of AChE. Interestingly, compound TM-9 abided by Lipinski's rule of five. Furthermore, our investigation proved that TM-9 indicated weak cytotoxicity, and it could cross the blood-brain barrier (BBB) in vitro. The results suggest that compound TM-9, an interesting multi-targeted active molecule, offers an attractive starting point for further lead optimization in the drug-discovery process against Alzheimer's disease.

Design, synthesis and biological evaluation of 2-acetyl-5-O-(amino-alkyl)phenol derivatives as multifunctional agents for the treatment of Alzheimer's disease.

A series of 2-acetyl-5-O-(amino-alkyl)phenol derivatives was designed, synthesized and evaluated as multi-function inhibitors for the treatment of Alzheimer's disease (AD). The results revealed that compound TM-3 indicated selective AChE inhibitory potency (eeAChE, IC50 = 0.69 µM, selective index (SI) = 32.7). Both kinetic analysis of AChE inhibition and molecular modeling study suggested that TM-3 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. And TM-3 was also a highly selective MAO-B inhibitor (IC50 = 6.8 µM). Moreover, TM-3 could act as antioxidant (ORAC value was 1.5eq) and neuroprotectant, as well as a selective metal chelating agent. More interestingly, compound TM-3 could cross the blood-brain barrier (BBB) in vitro and abided by Lipinski's rule of five. Therefore, compound TM-3, a promising multi-targeted active molecule, offers an attractive starting point for further lead optimization in the drug-discovery process against AD.

Downregulation of the long non-coding RNA TUG1 is associated with cell proliferation, migration, and invasion in breast cancer.

Recent studies have identified many long non-coding RNAs (lncRNAs) with critical roles in various biological processes including tumorigenesis. Taurine-upregulated gene 1 (TUG1), is an lncRNA recently reported to be involved in the progression of several human cancers. This study aimed to investigate the clinical significance and biological functions of TUG1 in breast cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure TUG1 expression in cells from breast cancer cell lines and in 58 matched pairs of breast cancer and normal tissue samples from patients with clinicopathological comparisons. Gain-and loss-of-function experiments were performed in vitro to investigate the biological role of TUG1. TUG1 expression was significantly downregulated in both breast cancer tissues and cell lines compared to controls, and low TUG1 expression was significantly correlated with mutant p53 expression (p=0.037) and lymph node metastasis (p=0.044). In vitro experiments revealed that TUG1 overexpression significantly suppressed cell proliferation by causing cell cycle arrest and inducing apoptosis in breast cancer cells, while TUG1 knockdown caused increased cell growth via promoting cell cycle progression and regulating the expression of cyclinD1 and CDK4. Further functional assays indicated that TUG1 overexpression significantly promoted cell migration and invasion while TUG1 knockdown had the opposite effects. Our findings indicate that the lncRNA TUG1 is a tumor suppressor in breast cancer, and may serve as a novel prognostic biomarker and potential therapeutic target for patients with breast cancer.

Design, synthesis and evaluation of novel ferulic acid-O-alkylamine derivatives as potential multifunctional agents for the treatment of Alzheimer's disease.

A series of novel ferulic acid-O-alkylamines derivatives were designed, synthesized, and evaluated as multitarget-directed ligands against Alzheimer's disease. In vitro studies displayed that all the synthesized target compounds showed impressive inhibitory activity against butyrylcholinesterase (BuChE), significant inhibition/disaggregation of self-induced ß-amyloid (Aß) aggregation and acted as potential antioxidants. Particularly, compound 7f, one of the most potent BuChE inhibitor (IC50 value of 0.021 µM for equine serum BuChE, 8.63 µM for ratBuChE and 0.07 µM for human serum BuChE), was found to be a good acetylcholinesterase (AChE) inhibitor (IC50 = 2.13 µM for electric eel AChE, 1.8 µM for ratAChE and 3.82 µM for human erythrocytes AChE), and the result of molecular docking provided an explanation for its selective BuChE inhibitory activity. Compound 7f also had noteworthy inhibitory effects on self-induced Aß1-42 aggregation (50.8 ± 0.82%) and was found to disaggregate self-induced Aß1-42 aggregation (38.7 ± 0.65%), which was further elucidated by the transmission electron microscopy. Meanwhile, compound 7f showed the modest antioxidant activity (0.55 eq of Trolox), good protective effect against H2O2-induced PC12 cell injury, with low toxicity. Moreover, compound 7f could cross the blood-brain barrier (BBB) in vitro. Significantly, compound 7f did not exhibit any acute toxicity in mice at doses up to 1000 mg/kg, and the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. Taken together, the results indicated that compound 7f is a very promising multifunctional agent in the treatment of Alzheimer's disease, particularly the advanced stages of AD.

Attempt towards a novel classification of triple-negative breast cancer using immunohistochemical markers.

Significant efforts have been made to gain a better understanding of the heterogeneity of triple-negative breast cancers from the histological to the molecular and genomic levels. In this study, we attempted to bring forward gene expression subtypes of triple-negative breast cancer (TBNC) to the clinic, by translating gene stratification to clinically accessible immunohistochemical (IHC) classification. Using IHC analysis, we categorized 154 TBNC cases into three main subclasses. Differences in the frequencies of basic characteristics and clinicopathological parameters between the subtypes were examined using Chi-square tests. We defined three main groups among the 154 triple-negative cases. The basal-like (BL) group expressed cytokeratin (CK) 5/6 and/or CK14 (83 cases), the AR+ group demonstrated positivity for androgen receptor (18 cases), and the final group exhibited a CD44+CD24-/low phenotype (39 cases). There were three overlapping cases between the BL subgroup and the CD44+CD24-/low phenotype subgroup, and 11 unclassified cases. In this new IHC classification, three subcategories exhibited a statistical difference with regard to age, tumor size, histological grade, tumor necrosis, Ki67 labeling index, relapse-free survival, breast cancer-specific survival and response to chemotherapy. According to our definition, the BL group and CD44+CD24-/low phenotype could be observed in tumors that were not triple-negative, and BL tumors that were triple-negative demonstrated almost undistinguishable clinicopathological characteristics compared with BL tumors that were not triple-negative. The same observation was made with CD44+CD24-/low tumors that were triple-negative vs. CD44+CD24-/low tumors that were not. The AR+ group demonstrated undistinguishable clinicopathological characteristics compared with the luminal subtype. We successfully distinguished three subtypes exhibiting diverse clinicopathological and prognostic characteristics with the minimum use of IHC markers.

Induction of autophagy by the MG­132 proteasome inhibitor is associated with endoplasmic reticulum stress in MCF­7 cells.

The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is involved in MG­132­induced autophagy, and to determine the effects of the inhibition of autophagy and ER stress on cell viability following MG­132 treatment. The proteasome inhibitor, MG­132, was used to induce autophagy in MCF­7 cells, and 3­methyladenine (3­MA) and salubrinal were used to inhibit autophagy and ER stress, respectively. An MTT assay was used to analyze cell viability. Apoptosis and the cell cycle were analyzed using flow cytometry. The expression levels of apoptosis­ and ER stress­associated genes were investigated using western blot and reverse transcription­quantitative polymerase chain reaction analyses. MG­132 inhibited cell proliferation, and induced apoptosis and cell cycle arrest at the G2 phase of the cell cycle. Notably, MG­132 increased the autophagy­associated conversion of microtubule­associated protein 1 light chain 3 (LC3)­I to LC3­II, which was partially attenuated by the ER stress inhibitor, salubrinal. In addition, MG­132 inhibited the protein expression of the anti­apoptotic protein, B­cell lymphoma (Bcl)­2, whereas the expression levels of Bcl­2­associated X protein and caspase­3 were upregulated. These effects were enhanced by co­treatment with either 3­MA or salubrinal. Furthermore, the mRNA and protein levels of the ER stress­associated genes, glucose­regulated protein 78, growth arrest and DNA damage induced gene­153, and caspase­12, were upregulated by MG132, and these levels were significantly inhibited by co­treatment of the cells with salubrinal. Taken together, the results of the present study indicated that the induction of autophagy by the proteasome inhibitor was associated with ER stress in the MCF­7 cells, and that the inhibition of autophagy or ER stress enhanced MG­132­induced apoptosis. These findings suggest the potential application of inhibitors of ER stress and autophagy, in combination with proteasomal inhibitors, for the development of combinatorial targeted cancer therapy.