Outcome of triple-negative breast cancer in patients with or without deleterious BRCA mutations.

More than 75% of breast cancers that develop in BRCA1 mutation carriers are triple-negative breast cancers (TNBC). The aim of this study was to compare the recurrence-free survival (RFS) and overall survival (OS) in high-risk patients with TNBC with and without deleterious BRCA1/2 mutations. A total of 227 women with TNBC who were referred for genetic counseling and underwent BRCA genetic testing between 1997 and 2010 were included in the study. The relationships between clinical variables and outcomes were evaluated using univariate and multivariate Cox proportional hazard regression models. Of 227 high-risk women with TNBC, 50% (n = 114) tested positive for BRCA1/2 mutations. Age, race, and tumor characteristics did not differ between BRCA non-carriers and carriers. At a median follow-up of 3.4 years, the 5-year RFS rates were 74 and 81% (P = 0.21), and 5-year OS rates were 85 and 93% in BRCA non-carriers and BRCA carriers, respectively (P = 0.11). In a multivariate model, after adjusting for age and disease stage, BRCA carriers tended to have a decreased risk of recurrence (HR = 0.67; 95% CI: 0.38-1.19; P = 0.17) or death (HR = 0.51; 95% CI:0.23-1.17; P = 0.11) compared to non-carriers. Our data indicate a 50% prevalence of deleterious BRCA1/2 mutations in high-risk women diagnosed with TNBC. Overall prognosis of TNBC in BRCA carriers and non-carriers is not significantly different within the first 5 years following an initial diagnosis. Further studies need to evaluate whether different therapies will change the outcome in these subgroups of TNBC.

Zyflamend mediates therapeutic induction of autophagy to apoptosis in melanoma cells.

Melanoma is the most aggressive form of skin cancer. The rising incidence of melanoma and its poor prognosis in advanced stages are compelling reasons to identify novel therapeutic agents. Though isolated dietary components such as lycopene, resveratrol, and isothiocyanate compounds have been shown to provide limited protection against cancer development, the use of whole herbs and herbal extracts for the treatment of cancer remains of great interest. As suggested by earlier studies, the antiinflammatory activity of many plants available as intact products or as extracts has long been considered for supplemental therapeutics for cancer. Zyflamend, a unique multiherbal extract preparation, is a promising antiinflammatory agent that has also been suggested to regulate multiple pathways in cancer progression. As Zyflamend contains ingredients that can suppress tumor cell proliferation, invasion, angiogenesis, and metastasis through regulation of inflammatory pathway products, we hypothesized that this preparation might inhibit melanoma proliferation. To test this hypothesis, we studied the effect of Zyflamend on melanoma proliferation. Here, we present that Zyflamend inhibits melanoma growth by regulating the autophagy-apoptosis switch. Based on the responsible molecular mechanisms of Zyflamend, our study highlights the importance of the use of herbal preparations for the prevention and treatment of cancer.

Autophagy is Required to Regulate Mitochondria Renewal, Cell Attachment, and All-trans-Retinoic Acid-Induced Differentiation in NB4 Acute Promyelocytic Leukemia Cells.

All-trans-retinoic acid (ATRA) is a potent inducer of cellular differentiation, growth arrest, and apoptosis as well as a front-line therapy for acute promyelocytic leukemia (APL). The present study provides evidence that induction of autophagy is required for ATRA to induce differentiation of APL (NB4) cells into granulocytes. ATRA treatment causes ~12-fold increase in the number of acidic vesicular organelles and induces marked up-regulation of LC3-II, autophagy-related 5 (ATG5), and Beclin-1. Transmission electron microscopy (TEM) revealed a decrease in mitochondria and ATRA-induced differentiation. To determine the role of autophagy in the differentiation of APL, we knocked down ATG5 in NB4 cells to find that ATRA-induced differentiation is significantly inhibited during ATG5 knock down in cells, indicating the role of autophagy in differentiation of APL. Further experiments revealed restriction of autophagy during ATRA-induced differentiation and inhibition of tissue transglutaminase 2 (TG2) and phospho-focal adhesion kinase (p-FAK), which are known to have roles in differentiation and cell attachment. We examined expression of Beclin-1 and B-cell lymphoma-2 (Bcl-2) and levels of mechanistic target of rapamycin (mTOR) after ATRA treatment. ATRA inhibits Bcl-2, up-regulates Beclin-1 expression, and reduces induction of mTOR activation/phosphorylation in NB4 cells. Our results reveal that autophagy has roles in regulation of differentiation, mitochondria elimination, and cell attachment during ATRA-induced APL differentiation.

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 microM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1-2 microM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97.

Tissue transglutaminase inhibits autophagy in pancreatic cancer cells.

Elevated expression of tissue transglutaminase (TG2) in cancer cells has been implicated in the development of drug resistance and metastatic phenotypes. However, the role and the mechanisms that regulate TG2 expression remain elusive. Here, we provide evidence that protein kinase Cdelta (PKCdelta) regulates TG2 expression, which in turn inhibits autophagy, a type II programmed cell death, in pancreatic cancer cells that are frequently insensitive to standard chemotherapeutic agents. Rottlerin, a PKCdelta-specific inhibitor, and PKCdelta small interfering RNA (siRNA) down-regulated the expression of TG2 mRNA and protein and induced growth inhibition without inducing apoptosis in pancreatic cancer cells. Inhibition of PKCdelta by rottlerin or knockdown of TG2 protein by a TG2-specific siRNA resulted in a marked increase in autophagy shown by presence of autophagic vacuoles in the cytoplasm, formation of the acidic vesicular organelles, membrane association of microtubule-associated protein 1 light chain 3 (LC3) with autophagosomes, and a marked induction of LC3-II protein, important hallmarks of autophagy, and by electron microscopy. Furthermore, inhibition of TG2 by rottlerin or by the siRNA led to accumulation of green fluorescent protein (GFP)-LC3-II in autophagosomes in pancreatic cancer cells transfected with GFP-LC3 (GFP-ATG8) expression vector. Knockdown of Beclin-1, a specific autophagy-promoting protein and the product of Becn1 (ATG6), inhibited rottlerin-induced and TG2 siRNA-induced autophagy, indicating that Beclin-1 is required for this process. These results revealed that PKCdelta plays a critical role in the expression of TG2, which in turn regulates autophagy. In conclusion, these results suggest a novel mechanism of regulation of TG2 and TG2-mediated autophagy in pancreatic cancer cells.

Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells.

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.

Polymorphisms of the XRCC1 DNA repair gene in head and neck cancer.

Inherited polymorphisms in the genes controlling the cell cycle or functioning in the DNA repair mechanisms may impair their function and contribute to genetic susceptibility. Abnormalities in the DNA repair have been reported in head and neck cancer. The XRCC1 gene functions in singlestrand break and base excision repair processes. In this study, two polymorphisms of the XRCC1 gene, Arg194Trp and Arg399Gln were investigated in 95 patients with head and neck carcinoma. The polymorphic regions were amplified by PCR followed by digestion with methylation-specific restriction enzymes, and analyzed electrophoretically. Genotype and allele frequencies were calculated, and association with cancer risk or clinical parameters was investigated. No association was observed between the genotypes and head and neck cancer for either polymorphism. Distribution of the alleles did not significantly differ between the patients and the control group. A significant association was only found for the Trp194 allele among the smoking individuals. Our data indicate that the Arg194Trp and Arg399Gln polymorphisms do not confer a significant risk for head and neck carcinogenesis.

The PARP inhibitor AZD2281 (Olaparib) induces autophagy/mitophagy in BRCA1 and BRCA2 mutant breast cancer cells.

PARP inhibitors are considered promising anticancer agents and currently being tested in clinical trials in hereditary breast cancer patients harboring mutations in BRCA1 and BRCA2 genes. In this study, we investigated the antiproliferative effects and mechanism of PARP inhibitors ABT-888 (Veliparib), BSI-201 (Iniparib) and AZD228 (Olaparib) in breast cancer cell lines with BRCA1 or BRCA2 mutations and 9 different BRCA wild-type cell lines with BRCA1 allelic loss. We found that AZD2281 was the most potent in the PARP inhibitors and induces significant growth inhibition (~95%) in BRCA1 mutant (HCC­1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC­1428) cell lines. AZD2281 treatment also resulted in growth inhibition ranging from 20 to 50% in cells with BRCA1 allelic loss, including ER(+), HER2/Neu(+) and triple-negative breast cancer (TNBC) cells, but showed no effect in cells without with type BRCA without allelic loss. Knocking down of BRCA1 or BRCA2 in TNBC cells with BRCA1 allelic loss by RNA interference significantly enhanced AZD2281-induced growth inhibition and induced significant autophagy that was associated with mitophagy in cells with BRCA mutations. Inhibition of autophagy by gene knockdown significantly diminished AZD2281-induced mitophagy and apoptosis, indicating that autophagic process mediates some of the downstream effects of PARP inhibitors. In conclusion, our data provide the first evidence of PARP inhibitor AZD2281 autophagy and mitophagy in breast cancer cell lines with BRCA mutations or BRCA-allelic loss. In addition, our results indicate that the patients with BRCA1 allelic loss may also benefit from PARP inhibitor therapy if BRCA is further inhibited.

[Venous thromboembolism prophylaxis with low molecular weight heparins in polytraumatized patients in intensive care unit (extended serie)]. / Politravmatize yogun bakim hastalarinda düsük molekül agirlikli heparin ile venöz tromboembolizm profilaksisi (genisletilmis seri).

BACKGROUND: In our study we aimed to evaluate, retrospectively, the bleeding and the Venousthrombo Emblism (VTE) complications in trauma patients in Intensive Care Unit (ICU) under the prophylaxis of enoxaparin sodium. The results will guide us to establish a protocol for use of Enoxaparin Sodium in trauma patients. In trauma patients, Low Molecular Weight Heparine (LMWH) has better efficiency than unfractionned heparin and intermittant pneumatic compression for VTE prophylaxis. METHODS: 457 polytraumatized patients treated in ICU are included to the study group. All patients received 40 mg/day of Enoxaparin Sodium. Severity of trauma was assessed with Apache II Scoring System and bleeding diagnosed by observing a sudden drop of 2 g/dl in hemoglobin concentration and pulmonary embolism was diagnosed by a sudden change in blood gases and deterioration of the clinical outlook which was confirmed by a spiral CT scan. Statistical correlation was made by Pearson's correlation test. RESULTS: Mean Apache II score was 13.8 with a total mortality of 41%. 42 patients (9,2%) had bleeding due to Enoxiparine Sodium prophylaxis. 12 patients (2,6%) had pulmonary embolism and 8 of them (1,7%) had died. CONCLUSION: Verified by the literature and our study, LMWH prophylaxis significantly reduces the Pulmoner Embolism (PE) incidence in polytraumatized patients (p<0,05). On the other hand, the bleeding risk has slightly increased without showing any significance (p>0.05). Prophylaxis with LMWH is safe and efficient in polytraumatized ICU patients.

Therapeutic Silencing of Bcl-2 by Systemically Administered siRNA Nanotherapeutics Inhibits Tumor Growth by Autophagy and Apoptosis and Enhances the Efficacy of Chemotherapy in Orthotopic Xenograft Models of ER (-) and ER (+) Breast Cancer.

Bcl-2 is overexpressed in about a half of human cancers and 50-70% of breast cancer patients, thereby conferring resistance to conventional therapies and making it an excellent therapeutic target. Small interfering RNA (siRNA) offers novel and powerful tools for specific gene silencing and molecularly targeted therapy. Here, we show that therapeutic silencing of Bcl-2 by systemically administered nanoliposomal (NL)-Bcl-2 siRNA (0.15 mg siRNA/kg, intravenous) twice a week leads to significant antitumor activity and suppression of growth in both estrogen receptor-negative (ER(-)) MDA-MB-231 and ER-positive (+) MCF7 breast tumors in orthotopic xenograft models (P < 0.05). A single intravenous injection of NL-Bcl-2-siRNA provided robust and persistent silencing of the target gene expression in xenograft tumors. NL-Bcl-2-siRNA treatment significantly increased the efficacy of chemotherapy when combined with doxorubicin in both MDA-MB-231 and MCF-7 animal models (P < 0.05). NL-Bcl-2-siRNA treatment-induced apoptosis and autophagic cell death, and inhibited cyclin D1, HIF1α and Src/Fak signaling in tumors. In conclusion, our data provide the first evidence that in vivo therapeutic targeting Bcl-2 by systemically administered nanoliposomal-siRNA significantly inhibits growth of both ER(-) and ER(+) breast tumors and enhances the efficacy of chemotherapy, suggesting that therapeutic silencing of Bcl-2 by siRNA is a viable approach in breast cancers.Molecular Therapy-Nucleic Acids (2013) 2, e121; doi:10.1038/mtna.2013.45; published online 10 September 2013.

PKCδ Regulates Translation Initiation through PKR and eIF2α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells.

Translation initiation and activity of eukaryotic initiation factor-alpha (eIF2α), the rate-limiting step of translation initiation, is often overactivated in malignant cells. Here, we investigated the regulation and role of eIF2α in acute promyelocytic (APL) and acute myeloid leukemia (AML) cells in response to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the front-line therapies in APL. ATRA and ATO induce Ser-51 phosphorylation (inactivation) of eIF2α, through the induction of protein kinase C delta (PKCδ) and PKR, but not other eIF2α kinases, such as GCN2 and PERK in APL (NB4) and AML cells (HL60, U937, and THP-1). Inhibition of eIF2α reduced the expression of cellular proteins that are involved in apoptosis (DAP5/p97), cell cycle (p21Waf1/Cip1), differentiation (TG2) and induced those regulating proliferation (c-myc) and survival (p70S6K). PI3K/Akt/mTOR pathway is involved in regulation of eIF2α through PKCδ/PKR axis. PKCδ and p-eIF2α protein expression levels revealed a significant association between the reduced levels of PKCδ (P = 0.0378) and peIF2 (P = 0.0041) and relapses in AML patients (n = 47). In conclusion, our study provides the first evidence that PKCδ regulates/inhibits eIF2α through induction of PKR in AML cells and reveals a novel signaling mechanism regulating translation initiation.

MEK1/2 inhibitor selumetinib (AZD6244) inhibits growth of ovarian clear cell carcinoma in a PEA-15-dependent manner in a mouse xenograft model.

Clear cell carcinoma (CCC) of the ovary tends to show resistance to standard chemotherapy, which results in poor survival for patients with CCC. Developing a novel therapeutic strategy is imperative to improve patient prognosis. Epidermal growth factor receptor (EGFR) is frequently expressed in epithelial ovarian cancer. One of the major downstream targets of the EGFR signaling cascade is extracellular signal-related kinase (ERK). PEA-15, a 15-kDa phosphoprotein, can sequester ERK in the cytoplasm. MEK1/2 plays a central role in integrating mitogenic signals into the ERK pathway. We tested the hypothesis that inhibition of the EGFR-ERK pathway suppresses tumorigenicity in CCC, and we investigated the role of PEA-15 in ERK-targeted therapy in CCC. We screened a panel of 4 CCC cell lines (RMG-I, SMOV-2, OVTOKO, and KOC-7c) and observed that the EGFR tyrosine kinase inhibitor erlotinib inhibited cell proliferation of EGFR-overexpressing CCC cell lines through partial dependence on the MEK/ERK pathway. Furthermore, erlotinib-sensitive cell lines were also sensitive to the MEK inhibitor selumetinib (AZD6244), which is under clinical development. Knockdown of PEA-15 expression resulted in reversal of selumetinib-sensitive cells to resistant cells, implying that PEA-15 contributes to selumetinib sensitivity. Both selumetinib and erlotinib significantly suppressed tumor growth (P < 0.0001) in a CCC xenograft model. However, selumetinib was better tolerated; erlotinib-treated mice exhibited significant toxic effects (marked weight loss and severe skin peeling) at high doses. Our findings indicate that the MEK-ERK pathway is a potential target for EGFR-overexpressing CCC and indicate that selumetinib and erlotinib are worth exploring as therapeutic agents for CCC.

Short-term biomarker modulation prevention study of anastrozole in women at increased risk for second primary breast cancer.

The selective estrogen receptor modulators (SERM), Tamoxifen and raloxifen reduce risk breast cancer. Patient acceptance of SERMs for breast cancer prevention is low due to toxicities. New agents with a better toxicity profile are needed. Aromatase inhibitors (AI) reduce the risk of contralateral breast cancer and risk of new breast cancer in high risk women. However, the mechanism by which AIs reduce breast risk is not known. Surrogate biomarkers are needed to evaluate the effect of preventive agents. The objective of this prospective short-term prevention study was to evaluate the effect of anastrozole on biomarkers in breast tissue and serum of women at increased risk for developing a contralateral breast cancer. Women with a history of stage I, II breast cancer who started anastrozole for standard adjuvant treatment were eligible. Patients underwent baseline fine needle aspiration of the unaffected breast and serum collection for biomarker analysis before starting anastrozole at 1 mg per oral/day and again at 6 months. Biomarkers included changes in cytology, insulin-like growth factor 1 (IGF-1), IGF-binding protein 1 (IGFBP-1), and IGFBP-3. Thirty-seven patients were enrolled. There was a significant modulation in serum IGFBP-1 levels between pre- and postsamples (P = 0.02). No change was observed in IGF-1, IGFBP-3, and breast cytology.We showed a significant modulation of IGFBP-1 levels with six months anastrozole. Anastrozole is currently being studied as a prevention agent in a large phase III trial and our results provide support for continued evaluation of IGFBP-1 as a surrogate endpoint biomarker in prospective breast chemoprevention studies.

Targeting p70S6K prevented lung metastasis in a breast cancer xenograft model.

Overexpression of p70S6K in breast cancer patients is associated with aggressive disease and poor prognosis. Recent studies showed that patients with breast cancer with increased p70S6K phosphorylation had poor survival and increased metastasis. The purpose of our study was to determine whether knockdown of p70S6K would inhibit cell growth, invasion, and metastasis in breast cancer. We therefore stably knocked down p70S6K expression in MDA-231, a highly metastatic breast cancer cell line, using a lentiviral short hairpin RNA (shRNA) based approach. Inhibition of p70S6K led to inhibition of cell growth, migration, and invasion in vitro. To determine the role of p70S6K in breast cancer tumorigenesis and metastasis, we used an MDA-231 orthotopic and metastatic animal model. In the orthotopic model, mice injected with MDA-231-p70S6K shRNA cells developed significantly smaller tumors than control mice injected with MDA-231 control shRNA cells (P < 0.01). No metastasis was observed in the p70S6K downregulated group, whereas lung metastasis was detected in all mice in the control group. To determine the role of p70S6K on growth and invasion, we tested downstream signaling targets by Western blot analysis. Knockdown of p70S6K inhibited phosphorylation of focal adhesion kinase, tissue transglutaminase 2, and cyclin D1 proteins, which promote cell growth, survival, and invasion. In addition, downregulation of p70S6K induced expression of PDCD4, a tumor-suppressor protein. In conclusion, we showed that p70S6K plays an important role in metastasis by regulating key proteins like cyclin D1, PDCD4, focal adhesion kinase, E-cadherin, beta-catenin, and tissue transglutaminase 2, which are essential for cell attachment, survival, invasion, and metastasis in breast cancer.

Silencing kinase-interacting stathmin gene enhances erlotinib sensitivity by inhibiting Ser¹° p27 phosphorylation in epidermal growth factor receptor-expressing breast cancer.

The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKI) such as erlotinib have been approved for cancer treatment but have shown very limited activity in breast cancer patients. Clarifying the molecular mechanism underlying resistance to EGFR TKIs could lead to more effective treatment against breast cancer. We previously reported that the sensitivity of breast cancer cells to erlotinib is partially dependent on p27 and that cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we found that erlotinib induces p27 phosphorylation at Ser¹° (S10), and S10 p27 phosphorylation leads to erlotinib resistance in EGFR-expressing breast cancer. Inhibiting S10 phosphorylation of p27 by knocking down human kinase-interacting stathmin (KIS), a nuclear protein that can phosphorylate p27 at S10, led to p27 accumulation in the nucleus and enhanced erlotinib-mediated cytotoxicity. Further, in vivo KIS gene silencing enhanced the antitumor activity of erlotinib in an orthotopic breast cancer xenograft model. KIS depletion also enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cells. Our study provides a rationale for the development of combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer.

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells.

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.

PKC delta and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells.

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKC delta) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKC delta/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKC delta/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.