Blockage of AMPK-ULK1 pathway mediated autophagy promotes cell apoptosis to increase doxorubicin sensitivity in breast cancer (BC) cells: an in vitro study.

BACKGROUND: Activation of autophagy flux contributed to resistance of breast cancer (BC) cells to current chemotherapeutic drugs, which seriously limited their therapeutic efficacy and facilitated BC recurrence in clinic. However, the detailed mechanisms are still not fully understood. In the present study, we identified that inactivation of AMPK-ULK1 signaling cascade mediated protective autophagy sensitized BC cells to doxorubicin in vitro. METHODS: Cell counting kit-8 (CCK-8) assay and colony formation assay were performed to evaluate cell proliferation abilities. Trypan blue staining assay was used to examine cell viability, and Annexin V-FITC/PI double staining method was conducted to determine cell apoptosis. The autophagosomes in BC cells were observed and photographed by electronic microscope (EM). Western Blot analysis was employed to examine genes expressions at protein levels. RESULTS: The parental doxorubicin-sensitive BC (DS-BC) cells were exposed to increasing concentrations of doxorubicin to establish doxorubicin-resistant BC (DR-BC) cells, and the DR-BC cells were much more resistant to high-dose doxorubicin treatment compared to the DS-BC cells. Interestingly, high-dose doxorubicin specifically increased LC3B-II/I ratio, promoted autophagosomes formation and decreased p62 expression levels to facilitate autophagy in DR-BC cells, instead of DS-BC cells, and the autophagy inhibitor 3-methyladenine (3-MA) enhanced the cytotoxic effects of high-dose doxorubicin on DR-BC cells. In addition, we proved that high-dose doxorubicin triggered protective autophagy in DR-BC cells by activating AMPK-ULK1 pathway. Functionally, high-dose doxorubicin increased the expression levels of phosphorylated AMPK (p-AMPK) and ULK1 (p-ULK1) to activate AMPK-ULK1 pathway in DR-BC cells, and the inhibitors for AMPK (compound C) and ULK1 (SBI-0206965) blocked autophagy to promote cell death and slow down cell growth in DR-BC cells treated with high-dose doxorubicin. CONCLUSIONS: Collectively, our in vitro data indicated that blockage of AMPK-ULK1 signaling cascade mediated protective autophagy might be a promising strategy to increase doxorubicin sensitivity for BC treatment.

LRH1 Promotes Tumor Cell Proliferation and Migration and Is Correlated With Poor Prognosis in Ovarian Cancer.

BACKGROUND: Liver receptor homolog 1 (LRH1) plays a vital role in several human cancers, but its role in ovarian cancer (OC) remains unclear. We aimed to explore the functions of LRH1 and its clinical relevance. METHODS: LRH1 expression was evaluated by immunohistochemistry and reverse transcription quantitative polymerase chain reaction (RT-qPCR). The effects of LRH1 on tumor cell proliferation, migration and epithelial-mesenchymal transition (EMT) were evaluated in vitro. Furthermore, bioinformatics analysis was applied to predict the functions of LRH1. RESULTS: RT-qPCR showed that LRH1 mRNA expression was higher in the invasive lesions (P < 0.05). LRH1 overexpression was extremely related with elevated International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.001), lymph node metastasis (P = 0.011), peritoneal metastasis (P = 0.001), and platinum resistance (P = 0.037). Furthermore, LRH1 expression was an independent prognostic index for disease-free survival in patients with OC (P = 0.041). LRH1 overexpression (P = 0.011), FIGO stage (P < 0.001), and ascites (P = 0.015) independently affected peritoneal metastasis in patients with OC. LRH1 knockdown significantly inhibited the proliferation, migration, and EMT of human OC cells (P < 0.05); however, it reversed cisplatin resistance. Bioinformatics analysis indicated that the functions of LRH1 were associated with the PRC1 complex, nuclear ubiquitin ligase complex, and Polycomb-group (PcG) proteins. CONCLUSIONS: This study provides evidence of the predictive value of LRH1 on peritoneal metastasis and poor outcome and highlights the potential role of LRH1 as a biomarker for the targeted therapy of OC. Furthermore, LRH1 promotes OC cell proliferation, migration, and EMT in vitro, and its functions may be associated with PRC1 complex, nuclear ubiquitin ligase complex, and PcG proteins.

Indications of clinical and genetic predictors for aromatase inhibitors related musculoskeletal adverse events in Chinese Han women with breast cancer.

BACKGROUND: Women with breast cancer treated with aromatase inhibitors (AIs) may experience musculoskeletal symptoms that lead to discontinuation of effective therapy. The purpose of the current study is to evaluate the clinical and genetic predictors for AIs-related musculoskeletal adverse events(MS-AEs). METHODOLOGY AND PRINCIPAL FINDINGS: We recruited 436 postmenopausal Chinese Han women receiving adjuvant AIs therapy for early-stage hormone-sensitive breast cancer. Patients completed a self-administered questionnaire assessing the presence of musculoskeletal symptoms that started or worsened after initiating AIs. 27 single nucleotide polymorphisms (SNP) of ESR1, ESR2 and PGR were analyzed by Sequenom MassARRAY assays and /or PCR-based TaqMan assays.Of the 436 enrolled women, 206 cases experienced musculoskeletal symptoms.Patients who received taxane chemotherapy were more than two times more likely than other patients to have AIs-related MS-AEs. Genetic assay had showed that only two ESR1 SNPs, rs2234693 and rs9340799 were associated with AIs-related MS-AEs.TT genotype and the T allele in rs2234693 was statistically significantly lower in AIs-Related MS-AEs group than controls (P = 0.001; P = 9.49E-7). The frequency of AA genotype and the A allele in rs9340799 was higher (P = 2.20E-5; P = 3.09E-4). CONCLUSIONS AND SIGNIFICANCE: Our results suggested that prior taxane-based chemotherapy was the clinical predictor, while rs2234693 and rs9340799 were the genetic predictors for AIs-related MS-AEs.

Analysis of 31 cases of primary breast lymphoma: the effect of nodal involvement and microvascular density.

BACKGROUND: We analyzed a database from our hospital comprised of 31 cases of primary breast lymphoma (PBL) that included treatment and follow-up information during the past 30 years, and investigated the correlation between microvessel density (MVD) and survival in patients with PBL. PATIENTS AND METHODS: We reviewed all patients diagnosed with primary breast lymphoma from June 1977 to March 2007. Patient demographics such as survival, recurrence, and time to follow-up were recorded, in addition to surgical, radiation, and/or chemotherapy treatment(s). We also assessed microvessel density (MVD) in the pretreatment breast lump of 31 previously untreated patients using α-CD34 immunohistochemical staining. RESULTS: All 31 patients were female ranging in age from 37 years to 75 years. Diffuse large B-cell lymphoma was the most common histologic diagnosis. According to the staging of Wiseman and Liao, 17 patients (55%) were stage IE, and 14 patients (45%) were stage IIE. Treatment that included radiation therapy in stage I patients (node negative) improve the survival rate and lowered the recurrence rates. Treatment that included chemotherapy in stage II patients (node positive) showed benefits in terms of higher survival rate and lower recurrence rate. Increasing microvessel density is a weak but statistically significant predictor of lower survival overall. CONCLUSION: Nodal status predicts the outcome and guides the use of radiation and chemotherapy. There is no statistical difference between the different types of operative methods. Patients with high MVD measured in the microenvironment had worse survival overall than PBL patients with low expression.

[Inhibitive effect of ursolic acid on the invasion and metastasis of ovarian carcinoma cells HO-8910PM].

OBJECTIVE: To study the effects of ursolic acid (UA) on the invasion and metastasis of ovarian carcinoma cell HO-8910PM and its underlying mechanism. METHODS: MTT assay was performed to examine the effects of UA on the proliferation of ovarian carcinoma cells HO-8910PM in vitro. The effects of UA on the invasion and migration of HO-8910PM were evaluated using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane. Gelatin zymography was performed to detect the activity of gelatinase in the HO-8910PM cells. The expressions of MMP-2 and MMP-9 in the HO-8910PM cells were measured by Western blot. RESULTS: UA inhibited the proliferation of HO-8910PM cells in a time- and dose-dependent manner. There was a statistically significant difference in the invasion and migration of HO-8910PM cells between the UA treated cells and the controls (P < 0.01, P < 0.05). UA inhibited the activity of gelatinase of the treated cells. CONCLUSION: UA downregulated the expressions of MMP-2 and MMP-9. UA inhibits the invasion and metastasis of HO-8910PM cells, probably through inhibiting the activity of gelatinase and the expressions of MMP-2 and MMP-9.

Malignant melanoma therapy by chemotherapy and autoimmunity induced by cytokine.

PURPOSE: The aim of this study was to evaluate the effect of combining dacarbazine (DTIC) and granulocyte/macrophage colony-stimulating factor (GM-CSF) with interleukin-2 (IL-2) in patients with advanced malignant melanoma. METHODS: Twenty-seven (27) patients with advanced malignant melanoma received dacarbazine (500 mg/m(2)/days 1-2, intravenously), GM-CSF (175 ug/m(2)/days 3-6, subcutaneously), and interleukin-2 (400 MIU/m(2)/days 7-10, subcutaneously). Each treatment cycle required 21 days to completion. RESULTS: Time to progression was 7-11 months. The total effective rate was 44.4%, and the combination of chemotherapy, GM-CSF, and IL-2 had low toxicity. CONCLUSIONS: The combination of DTIC with GM-CSF and IL-2 is feasible and possibly efficacious for clinical use.

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T.

AIM: To investigate the effect and mechanism of action of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS: Human colorectal cancer cell line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h. Nitric oxide (NO) production was measured with Griess reagent. The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel). RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor metalloproteinase-2 (TIMP-2). RESULTS: L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L-NAME, respectively, the ability of the L-NAME treated SL-174T cells to invade the reconstituted basement membrane decreased significantly (t = 8.056, P< 0.05; t = 14.467, P< 0.01; t = 27.785, P< 0.01; and t = 29.405, P< 0.01, respectively) and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46.85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively (t= 15.116, P< 0.01). In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased (t = 71.238, P< 0.01) and that of TIMP-2 mRNA was markedly increased (t = -13.020, P< 0.01). CONCLUSION: L-NAME exerts anti-invasive and anti-metastatic effects on SL-174T cell line via downregulating MMP-2 mRNA expression and upregulating TIMP-2 mRNA expression.