In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-microM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFkappaB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFkappaB activation and then decreases cell proliferation.
Antineoplastic Agents, Alkylating/*pharmacology ; Breast Neoplasms/drug therapy/enzymology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Diterpenes/*pharmacology ; Epoxy Compounds/pharmacology ; Female ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; NF-kappa B/genetics/metabolism ; Phenanthrenes/*pharmacology ; Phospholipase D/*genetics/metabolism

Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays an important role in tumorigenesis. Whether Ubc9 is involved in the chemoresistance of breast cancer remains unknown. In this study, we aimed to evaluate the contribution of Ubc9 in the chemoresistance of breast cancer. Immunohistochemistry (IHC) was used to examine the expression level of Ubc9. Chi-square test, Wilcoxon test, and one-way ANOVA were applied to analyze the relationship between Ubc9 expression, clinicopathologic features, and clinical response to neoadjuvant chemotherapy. The significance of variables for survival was analyzed by the Cox proportional hazards model in a multivariate analysis. Kaplan-Meier survival curves were plotted and log-rank test was performed. The proportion of Ubc9-positive cells was higher in invasive ductal carcinoma than in normal breast tissues [(48.48 ± 17.94)% vs. (5.82 ± 2.80)%, P < 0.001]. High Ubc9 expression was associated with poor differentiation (Χ² = 6.538, P = 0.038), larger tumor size (Χ² = 4.701, P = 0.030), advanced clinical stage (Χ² = 4.651, P = 0.031), lymph node metastasis (Χ² = 9.913, P = 0.010), basal-like phenotype (Χ² = 8.660, P = 0.034), and poor clinical response to neoadjuvant chemotherapy (Χ² = 11.09, P = 0.001). The expected 6-year cumulative disease-free survival rate was 87.32% in patients with low Ubc9 expression compared to 68.78% in those with high Ubc9 expression (Χ² = 4.289, P = 0.038). These data indicate that high Ubc9 expression correlates with poor response to chemotherapy and poor clinical prognosis.
Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; enzymology ; pathology ; surgery ; Carcinoma, Ductal, Breast ; drug therapy ; enzymology ; pathology ; surgery ; Cyclophosphamide ; therapeutic use ; Disease Progression ; Disease-Free Survival ; Drug Resistance, Neoplasm ; Epirubicin ; therapeutic use ; Female ; Fluorouracil ; therapeutic use ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Mastectomy ; methods ; Middle Aged ; Neoadjuvant Therapy ; Neoplasm Staging ; Proportional Hazards Models ; Remission Induction ; Tumor Burden ; Ubiquitin-Conjugating Enzymes ; metabolism ; Up-Regulation

OBJECTIVETo investigate the expression of extracellular signal-regulated kinase (ERK) and its relationship with clinicopathological characteristics of breast cancer as well as the effect of preoperative chemotherapy on ERK expression.

METHODSExpression of ERK-1 and ERK-2 protein was examined by Western blot in the breast cancer and normal breast (control) tissue of 48 patients, of whom 8 had received preoperative chemotherapy of 5'-deoxy-5-fluorouridine (5'-DFUR), with distribution of ERKs protein detected by immunohistochemical method.

RESULTSExpression of ERK-1 and ERK-2 protein was increased in tumor specimen as compared with control tissue (P < 0.01). A positive correlation was observed between ERK-1 and ERK-2 (r = 0.457, P < 0.01). Protein level of ERK-1 and ERK-2 was higher in stage III patients than in stage I and stage II patients (P < 0.05). Expression of both ERK-1 and ERK-2 in the carcinoma tissue was decreased in patients who had received preoperative chemotherapy of 5'-DFUR. ERK-1 and ERK-2 proteins were mainly located in the cytoplasm.

CONCLUSIONThe hyperexpression of ERK may play an important role in the initiation and development of human breast cancer. Preoperative chemotherapy of 5'-DFUR is able to partially inhibit ERK expression.

Antimetabolites, Antineoplastic ; therapeutic use ; Breast Neoplasms ; classification ; drug therapy ; enzymology ; pathology ; Female ; Floxuridine ; therapeutic use ; Humans ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; biosynthesis ; Neoplasm Staging

PARP is an important protein in DNA repair pathways especially the base excision repair (BER). BER is involved in DNA repair of single strand breaks (SSBs). If BER is impaired, inhibiting poly(ADP-ribose) polymerase (PARP), SSBs accumulate and become double stand breaks (DSBs). The cells with increasing number of DSBs become more dependent on other repair pathways, mainly the homologous recombination (HR) and the nonhomologous end joining. Patients with defective HR, like BRCA-deficient cell lines, are even more susceptible to impairment of the BER pathway. Inhibitors of PARP preferentially kill cancer cells in BRCA-mutation cancer cell lines over normal cells. Also, PARP inhibitors increase cytotoxicity by inhibiting repair in the presence of chemotherapies that induces SSBs. These two principles have been tested clinically. Over the last few years, excitement over this class of agents has escalated due to reported activity as single agent in BRCA1- or BRCA2-associated ovarian or breast cancers, and in combination with chemotherapy in triple negative breast cancer. This review covers the current results of clinical trials testing those two principles. It also evaluates future directions for the field of PARP inhibitor development.
Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Benzamides ; administration & dosage ; Benzimidazoles ; administration & dosage ; Breast Neoplasms ; drug therapy ; enzymology ; genetics ; DNA Breaks, Double-Stranded ; DNA Breaks, Single-Stranded ; DNA End-Joining Repair ; DNA Repair ; Enzyme Inhibitors ; therapeutic use ; Female ; Genes, BRCA1 ; Genes, BRCA2 ; Homologous Recombination ; Humans ; Mutation ; Ovarian Neoplasms ; drug therapy ; enzymology ; genetics ; Phthalazines ; administration & dosage ; Piperazines ; administration & dosage ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism

27-O-(E)-p-coumaric acyl ursolic acid( DY-17) from Ilex latifolia is a compound of the monomer. To investigate the DY-17 inducing apoptosis in the human breast cancer cell line, the MDA-MB-231 cells were used as research object in this experiment. The proliferation activity of the MDA-MB-231 cells stimulated with the different concentrations of DY-17 (20, 40 µmol · L(-1)) was detected at different time( 12, 24, 36, 48, 60,72 h) . We surveyed the DY-17 inducing apoptosis of the MDA-MB-231 cells with the fluorescent staining technology. The rate of MDA-MB-231 cells apoptosis and necrosis was determined by flow cell cytometry (FCC). Moreover, expression of JNK, phosphorylated JNK, Bax, PARP shear and caspase-3 shear related to JNK/SAPK pathways were investigated in every group ( control group, EGF group, EGF + DY-17 40 µmol · L(1) group and EGF + SP600125 group) with Western blot. The MTT results showed that, in the presence of DY-17, the proliferation activity of MDA-MB-231 cells decreased in a dose-dependent and time-dependent manner. The apoptosis and necrosis rates of MDA-MB-231 cells with DY-17(20, 40 µmol · L(-1)) groups was respectively 31.86%, 49.91% by flow cytometry and significantly increased compared with control group under Fluores- cence microscopy. Up-regulation of the JNK phosphorylation protein expression was observed in EGF group compared with control group. In addition, markedly decreased the expression of JNK phosphorylation protein were also surveyed in EGF + DY-17 40 µmol · L(-1) group compared with EGF group. The expression of Bax, shear PARP and shear caspase-3 protein in EGF + DY-17 40 µmol · L(-1) group were significantly increased in comparison with EGF group. The results showed DY-17 induced apoptosis of human breast cancer MDA-MB-231 cell line related to down-regulating JNK/SAPK signal pathways.
Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; enzymology ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; MAP Kinase Kinase 4 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Signal Transduction ; drug effects ; Triterpenes ; chemistry ; pharmacology

To study the effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis and its possible mechanism. MTT assay was used to detect the inhibitory effect of sodium aescinate on the proliferation of MCF-7 cells. The morphological changes were observed under inverted microscope. DAPI nuclear staining was used to detect the changes in cell nucleus. Annexin V-FITC/PI flow cytometry was adopted to test the apoptosis rate. Changes in apoptosis-related proteins (PARP, cleaved caspase-8 and pro-caspase-3), cell survival-associated signal molecules (AKT and ERK) and their common upstream kinase SRC was detected by Western blotting. The result showed that after different concentrations of sodium aescinate were used to treat breast cancer MCF-7 cells, they inhibited the proliferation of MCF-7 cells in a dose-dependent manner, induced cell apoptosis (typical morphological changes in nucleus, significant increase in cell apoptosis rate). The expressions of cleaved PARP and caspase-8 increased, while the expression of pro-caspase-3 decreased, which further verified sodium aescinate's effect in inducing cell apoptosis. Sodium aescinate significantly inhibited the phosphorylation of cell survival-related signal molecules (AKT, ERK) and down-regulate the activation of their common up-stream kinase SRC. The findings indicated that sodium aescinate can block signals transiting to downstream molecules AKT, ERK, inhibit the proliferation of breast cancer cell MCF-7 cell apoptosis and induced cell apoptosis by suppressing the activation of SRC.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; drug therapy ; enzymology ; genetics ; physiopathology ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Female ; Humans ; MCF-7 Cells ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Triterpenes ; pharmacology ; src-Family Kinases ; genetics ; metabolism

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.
Breast Neoplasms/*drug therapy/*enzymology/pathology ; Cell Death/drug effects/radiation effects ; Cell Line, Tumor ; Cell Proliferation/drug effects/radiation effects ; DNA Damage ; Enzyme Activation/drug effects/radiation effects ; Enzyme Inhibitors/*pharmacology/*therapeutic use ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Female ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Phospholipase D/*antagonists & inhibitors/metabolism ; Radiation Tolerance/*drug effects ; Radiation, Ionizing ; p38 Mitogen-Activated Protein Kinases/metabolism