Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms
associated with enhancing the activity of lapatinib via combination with other therapies.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
A new assay for measuring chromosome instability (CIN) and identification of...Enrique Moreno Gonzalez
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the
CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory.
CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1a...Enrique Moreno Gonzalez
Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.
Abnormal expression of Pygopus 2 correlates with a malignant phenotype in hum...Enrique Moreno Gonzalez
Pygopus 2 (Pygo2) is a Pygo family member and an important component of the Wnt signaling transcriptional complex. Despite this data, no clinical studies investigating Pygo2 expression in lung cancer have yet been reported.
Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated ...Enrique Moreno Gonzalez
Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants,
vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17β-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August
Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis.
ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grad...Enrique Moreno Gonzalez
High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is
mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas.
Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed highgrade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients.
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
Search for atoxic cereals: a single blind, cross-over study on the safety of...Enrique Moreno Gonzalez
Cereals of baking quality with absent or reduced toxicity are actively sought as alternative therapy to a gluten-free diet (GFD) for patients with coeliac disease (CD). Triticum monococcum, an ancient wheat, is a potential candidate having no toxicity in in-vitro and exvivo studies. The aim of our study was to investigate on the safety of administration of a single dose of gluten of Tm in patients with CD on GFD.
A new assay for measuring chromosome instability (CIN) and identification of...Enrique Moreno Gonzalez
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the
CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory.
CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1a...Enrique Moreno Gonzalez
Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion.
Abnormal expression of Pygopus 2 correlates with a malignant phenotype in hum...Enrique Moreno Gonzalez
Pygopus 2 (Pygo2) is a Pygo family member and an important component of the Wnt signaling transcriptional complex. Despite this data, no clinical studies investigating Pygo2 expression in lung cancer have yet been reported.
Lack of association between CD45 C77G polymorphism and multiple sclerosis in ...ijtsrd
Multiple sclerosis (MS) is a severe disabling and demyelinating disease of the nervous system. Its etiology involves profound genetic component. The latest contender known to have been correlated with MS is protein tyrosine phosphatase receptor-type C (PTPRC or CD45); however, to date its role remains contentious. The aim of the current study was to examine the association of functionally significant exon 4 C77G polymorphism of CD45 with MS in Kashmiri population from Indian subcontinent. The preliminary findings of our study revealed absence of C77G in majority of the cases as well as controls. These findings strongly suggest that the alterations in CD45 are sporadically associated with the genesis of MS. In conclusion, results from our study are in accordance with some of the international studies; however, more studies with large datasets from Kashmir as well as other ethnic populations are warranted to validate the above preliminary findings and demonstrate the role of CD45 C77G polymorphism in MS pathogenesis. Insha Zahoor | Amrina Shafi | Mudasir A Mir"Lack of association between CD45 C77G polymorphism and multiple sclerosis in Kashmir" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-1 | Issue-6 , October 2017, URL: http://www.ijtsrd.com/papers/ijtsrd5813.pdf http://www.ijtsrd.com/biological-science/biotechnology/5813/lack-of-association-between-cd45-c77g-polymorphism-and--multiple-sclerosis-in-kashmir/insha-zahoor
Functional p53 is required for rapid restoration of daunorubicin-induced lesi...Enrique Moreno Gonzalez
The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen.
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
The Effects of Genetic Alteration on Reprogramming of Fibroblasts into Induc...remedypublications2
Induced Pluripotent Stem Cells (iPSCs) can be generated from somatic cells by ectopic expression of
Yamanaka factors (
Oct4
,
Sox2
,
Klf4
and
c-Myc
) or combination of other factors. Genetic alteration
of fibroblasts exhibits an effect on reprogramming efficiency through multiple signaling pathways,
including epigenetic modifications, metabolic shifts, Mesenchymal-To-Epithelial Transition
(MET) and cell proliferation. In order to better understand the underlying mechanisms in cell fate
determination, in this review we will summarize several genetic alterations involved in the regulation
of reprogramming fibroblasts into iPSCs.
Immunotherapy: Novel Immunomodulatory TargetsPaul D. Rennert
An approach to discovering new immunotherapy targets for oncology is introduced and examples presented. New programs from biotech and pharma are discussed.
Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.
Multicentric and multifocal versus unifocal breast cancer: differences in the...Enrique Moreno Gonzalez
The aim of this study was to evaluate the expression of the cell adhesion-related glycoproteins MUC-1, β-catenin and E-cadherin in multicentric/multifocal breast cancer in comparison to unifocal disease in order to identify potential differences in the biology of these tumor types.
Lack of association between CD45 C77G polymorphism and multiple sclerosis in ...ijtsrd
Multiple sclerosis (MS) is a severe disabling and demyelinating disease of the nervous system. Its etiology involves profound genetic component. The latest contender known to have been correlated with MS is protein tyrosine phosphatase receptor-type C (PTPRC or CD45); however, to date its role remains contentious. The aim of the current study was to examine the association of functionally significant exon 4 C77G polymorphism of CD45 with MS in Kashmiri population from Indian subcontinent. The preliminary findings of our study revealed absence of C77G in majority of the cases as well as controls. These findings strongly suggest that the alterations in CD45 are sporadically associated with the genesis of MS. In conclusion, results from our study are in accordance with some of the international studies; however, more studies with large datasets from Kashmir as well as other ethnic populations are warranted to validate the above preliminary findings and demonstrate the role of CD45 C77G polymorphism in MS pathogenesis. Insha Zahoor | Amrina Shafi | Mudasir A Mir"Lack of association between CD45 C77G polymorphism and multiple sclerosis in Kashmir" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-1 | Issue-6 , October 2017, URL: http://www.ijtsrd.com/papers/ijtsrd5813.pdf http://www.ijtsrd.com/biological-science/biotechnology/5813/lack-of-association-between-cd45-c77g-polymorphism-and--multiple-sclerosis-in-kashmir/insha-zahoor
Functional p53 is required for rapid restoration of daunorubicin-induced lesi...Enrique Moreno Gonzalez
The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen.
Molecular mechanisms of action and potential biomarkers of growth inhibition ...Enrique Moreno Gonzalez
Molecular targeted therapy has emerged as a promising treatment of Hepatocellular carcinoma (HCC). One potential target is the Src family Kinase (SFK). C-Src, a non-receptor tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, survival, metastasis, and angiogenesis. In this study, we evaluated the effects of a novel SFK inhibitor, dasatinib (BMS-354825), on SFK/FAK/p130CAS, PI3K/PTEN/Akt/mTOR, Ras/Raf/MAPK and Stats pathways in 9 HCC cell lines.
The Effects of Genetic Alteration on Reprogramming of Fibroblasts into Induc...remedypublications2
Induced Pluripotent Stem Cells (iPSCs) can be generated from somatic cells by ectopic expression of
Yamanaka factors (
Oct4
,
Sox2
,
Klf4
and
c-Myc
) or combination of other factors. Genetic alteration
of fibroblasts exhibits an effect on reprogramming efficiency through multiple signaling pathways,
including epigenetic modifications, metabolic shifts, Mesenchymal-To-Epithelial Transition
(MET) and cell proliferation. In order to better understand the underlying mechanisms in cell fate
determination, in this review we will summarize several genetic alterations involved in the regulation
of reprogramming fibroblasts into iPSCs.
Immunotherapy: Novel Immunomodulatory TargetsPaul D. Rennert
An approach to discovering new immunotherapy targets for oncology is introduced and examples presented. New programs from biotech and pharma are discussed.
Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.
Multicentric and multifocal versus unifocal breast cancer: differences in the...Enrique Moreno Gonzalez
The aim of this study was to evaluate the expression of the cell adhesion-related glycoproteins MUC-1, β-catenin and E-cadherin in multicentric/multifocal breast cancer in comparison to unifocal disease in order to identify potential differences in the biology of these tumor types.
A phase I/II trial to evaluate the safety, feasibility and activity of salvag...Enrique Moreno Gonzalez
The current standard treatment of patients with relapsed or refractory diffuse large cell B-Cell lymphoma (DLBCL) primarily consists of intensified salvage therapy and, if the disease is chemo-sensitive, high dose therapy followed with autologous stem cell transplantation. In the rituximab era however, this treatment approach has shown only limited benefit. In particular, patients relapsing after rituximab-containing primary treatment have an adverse prognosis, especially if this occurs within the first year after therapy or if the disease is primarily refractory. Therefore there is an ultimate need for improved salvage treatment approaches.
Association between variations in the fat mass and obesity-associated gene an...Enrique Moreno Gonzalez
It is clear that genetic variations in the fat mass and obesity-associated (FTO) gene affect body mass index and the risk of obesity. Given the mounting evidence showing a positive association between obesity and pancreatic cancer, this study aimed to investigate the relation between variants in the FTO gene, obesity and pancreatic cancer risk.
Intraepithelial lymphocyte distribution differs between the bulb and the seco...Enrique Moreno Gonzalez
Evaluation of intraepithelial duodenal lymphocytosis (IDL) is important in celiac disease (CD). There is no established cut-off value for increased number of IELs in the bulb. We therefore investigated the relation between IEL counts in the bulb and duodenal specimens in non-celiac subjects.
Implication from thyroid function decreasing during chemotherapy in breast ca...Enrique Moreno Gonzalez
Thyroid hormones have been shown to regulate breast cancer cells growth, the absence or reduction of thyroid hormones in cells could provoke a proliferation arrest in G0-G1 or weak mitochondrial activity, which makes cells insensitive to therapies for cancers through transforming into low metabolism status. This biological phenomenon may help explain why treatment efficacy and prognosis vary among breast cancer patients having hypothyroid, hyperthyroid and normal function. Nevertheless, the abnormal thyroid function in breast cancer patients has been considered being mainly caused by thyroid diseases, few studied influence of chemotherapy on thyroid function and whether its alteration during chemotherapy can influence the respose to chemotherapy is still unclear. So, we aimed to find the alterations of thyroid function and non-thyroidal illness syndrome (NTIS) prevalence druing chemotherapy in breast cancer patients, and investigate the influence of thyroid hormones on chemotherapeutic efficacy.
Assessment of preoperative exercise capacity in hepatocellular carcinoma pati...Enrique Moreno Gonzalez
Cardiopulmonary exercise testing measures oxygen uptake at increasing levels of work and predicts cardiopulmonary performance under conditions of stress, such as after abdominal surgery. Dynamic assessment of preoperative exercise capacity may be a useful predictor of postoperative prognosis. This study examined the relationship between preoperative exercise capacity and event-free survival in hepatocellular carcinoma (HCC) patients with chronic liver injury who underwent hepatectomy.
Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FO...Enrique Moreno Gonzalez
Although advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function.
Inhibition of glutathione by buthionine sulfoximine enhanced the anti-cancer ...Ashujit
Multiple myeloma (MM) is an incurable blood cancer. Melphalan is an alkylating agent given prior to stem cell transplantation to MM patients. Increased glutathione confers resistance to melphalan. This study investigate the effect of inhibition of glutathione by BSO in preclinical models of MM. Pretreatment with BSO enhanced the anti-cancer effect of melphalan in cell lines and animal models. BSO and melphalan combination was well tolerated by animals and enhanced the survival as compared to controls, BSO and melphalan alone. BSO enhanced depth and duration of responses induced by melphalan. In the combination group, majority of treated animals achieved complete response (CR) and more than 20% had maintained CR. Also, the survival of animals was doubled after combination treatment as compared to BSO or melphalan alone. Mechanistic investigation demonstrated that BSO enhanced melphalan induced DNA damage, caspase cleavage and apoptosis. The combination also achieved multi-logs of cells kills in nine human multiple myeloma cell lines and primary MM cells isolated from blood and bone marrows. Interestingly, the effect of BSO and melphalan combination was abolished when cells were treated with N-acetyl cysteine and sodium thiosulfate but not with vitamin C and vitamin E. This observation suggests that effect of BSO is primarily driven by its ability to deplete glutathione and therefore preventing melphalan detoxification. Together, this study provides framework for testing the combination in a Phase I trial.
Keto reductases (AKRs) are overexpressed in a large number of human tumors and mediate
resistance to cancer chemotherapeutics and antihormonal therapies. Existing drugs and new agents in development may surmount this resistance by acting as specific AKR isoforms or AKR
pan-inhibitors to improve clinical outcome.
Keto reductases (AKRs) are overexpressed in a large number of human tumors and mediate
resistance to cancer chemotherapeutics and antihormonal therapies. Existing drugs and new
agents in development may surmount this resistance by acting as specific AKR isoforms or AKR
pan-inhibitors to improve clinical outcome.
Keto reductases (AKRs) catalyze the NADPH-dependent reduction of carbonyl groups to
alcohols for conjugation reactions to proceed. They are implicated in resistance to cancer
chemotherapeutic agents either because they are directly involved in their metabolism or help
eradicate the cellular stress created by these agents (e.g., reactive oxygen species and lipid
peroxides). Furthermore, this cellular stress activates the nuclear factor-erythroid 2 p45-related
factor 2 (NRF2)-Kelch-like ECH-associated protein 1 pathway. As many human AKR genes are
upregulated by the NRF2 transcription factor, this leads to a feed-forward mechanism to enhance
drug resistance. Resistance to major classes of chemotherapeutic agents (anthracyclines,
mitomycin, cis-platin, antitubulin agents, vinca alkaloids, and cyclophosphamide) occurs by this
mechanism. Human AKRs also catalyze the synthesis of androgens and estrogens and the
elimination of progestogens and are involved in hormonal-dependent malignancies. They are
upregulated by antihormonal therapy providing a second mechanism for cancer drug resistance.
Inhibitors of the NRF2 system or pan-AKR1C inhibitors offer promise to surmount cancer drug
resistance and/or synergize the effects of existing drugs.
Integrative Medicine and Experimental Pharmacogenomic Therapy in a Child with...Sophia's Garden Foundation
A 34-month female with Niemann-Pick Disease, Type A (NPD-A) is presented. NPD-A is a rare autosomal recessive, hereditary lysosomal storage disease caused by reduced acid sphingomyelinase (ASM) activity. Reduced sphingomyelinase activity resulting in impaired sphingomyelin turnover leads to cellular lipid and lysosomal storage (foam cells), hepatosplenomegaly, delay and loss of developmental milestones, and degenerative neurologic deficits. Death occurs in infancy or early childhood, with the usual life span being 2-3 years. No effective treatment for NPD-A is known, although bone marrow transplantation and enzyme replacement therapy with ASM have been attempted. [Both have been tried for NPD-B, but I do not know if either of these techniques have been tried for NPD-A. This is true that no successful treatment has been demonstrated for humans with NPD-A, yet the Genzyme studies using adeno viruses were successful for mice.
See <http://www.genzyme.co.uk/corp/news/all_news/GENL%20PR-060503.asp>. Stem cell and gene therapy research is being investigated in animal models. This child is known to have an unusual stop-codon mutation. Aminoglycosides have been found in animal and small-scale human studies to cause read-through in translation effectively skipping the “stop-codon” and are in clinical trials in other genetic diseases with similar mutations. It was hypothesized that gentamicin would cause read-through in translation in this patient, and thereby increase production of acid sphingomyelinase. After presentation to the Ethics Committee of Lucile Salter Packard Children’s Hospital at Stanford University, the child was treated with an experimental protocol using intranasal gentamicin. This is the first case of NPD-A in which this has been tried.
This patient has also been treated with Traditional Chinese Medicine including acupuncture and botanicals, massage therapy, nutritional measures, homeopathy, energy healing and compassionate intention in an Integrative Medicine approach. Disease characteristics, clinical features and course will be described.
Incidence of pneumonia and risk factors among patients with head and neck can...Enrique Moreno Gonzalez
This study investigated the incidence and patient- and treatment-related risk factors related to pneumonia acquired during radiotherapy (PNRT) in head and neck cancer (HNC) patients.
Gene expression analysis of a Helicobacter pyloriinfected and high-salt diet-...Enrique Moreno Gonzalez
Helicobacter pylori (H. pylori) infection and excessive salt intake are known as important risk factors for stomach cancer in humans. However, interactions of these two factors with gene expression profiles during gastric carcinogenesis remain unclear. In the present study, we investigated the global gene expression associated with stomach carcinogenesis and prognosis of human gastric cancer using a mouse model.
Recently, a phase II clinical trial in hepatocellular carcinoma (HCC) has suggested that the combination of sorafenib and 5-fluorouracil (5-FU) is feasible and side effects are manageable. However, preclinical experimental data explaining the interaction mechanism(s) are lacking. Our objective is to investigate the anticancer efficacy and mechanism of combined sorafenib and 5-FU therapy in vitro in HCC cell lines MHCC97H and SMMC-7721.
Differences in microRNA expression during tumor development in the transition...Enrique Moreno Gonzalez
The prostate is divided into three glandular zones, the peripheral zone (PZ), the transition zone (TZ), and the central zone. Most prostate tumors arise in the peripheral zone (70-75%) and in the transition zone (20-25%) while only 10% arise in the central zone. The aim of this study was to investigate if differences in miRNA expression could be a possible explanation for the difference in propensity of tumors in the zones of the prostate.
The life in sight application study (LISA): design of a randomized controlled...Enrique Moreno Gonzalez
It is widely recognized that spiritual care plays an important role in physical and psychosocial well-being of cancer patients, but there is little evidence based research on the effects of spiritual care. We will conduct a randomized controlled trial on spiritual care using a brief structured interview scheme supported by an e-application. The aim is to examine whether an assisted reflection on life events and ultimate life goals can improve quality of life of cancer patients.
Clinical and experimental studies regarding the expression and diagnostic val...Enrique Moreno Gonzalez
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a multifunctional Ig-like cell adhesion molecule that has a wide range of biological functions. According to previous reports, serum CEACAM1 is dysregulated in different malignant tumours and associated with tumour progression. However, the serum CEACAM1 expression in nonsmall-cell lung carcinomas (NSCLC) is unclear. The different expression ratio of CEACAM1-S and CEACAM1-L isoform has seldom been investigated in NSCLC. This research is intended to study the serum CEACAM1 and the ratio of CEACAM1-S/L isoforms in NSCLC.
Overexpression of YAP 1 contributes to progressive features and poor prognosi...Enrique Moreno Gonzalez
Yes-associated protein 1 (YAP 1), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene in multiple tumors. However, the expression dynamics of YAP 1 in urothelial carcinoma of the bladder (UCB) and its clinical/prognostic significance are unclear.
Differentiation of irradiation and cetuximab induced skin reactions in patien...Enrique Moreno Gonzalez
In order to improve the clinical outcome of patients with locally advanced squamous cell carcinoma of the head and neck (LASCCHN) not being capable to receive platinum-based chemoradiation, radiotherapy can be intensified by addition of cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR). The radioimmunotherapy with cetuximab is a feasible treatment option showing a favourable toxicity profile. The most frequent side effect of radiotherapy is radiation dermatitis, the most common side effect of treatment with cetuximab is acneiform rash. Incidence and severity of these frequent, often overlapping and sometimes limiting skin reactions, however, are not well explored. A clinical and molecular differentiation between radiogenic skin reactions and skin reactions caused by cetuximab which may correlate with outcome, have never been described before.
Cholestasis induces reversible accumulation of periplakin in mouse liverEnrique Moreno Gonzalez
Periplakin (PPL) is a rod-shaped cytolinker protein thought to connect cellular adhesion junctional complexes to cytoskeletal filaments. PPL serves as a structural component of the cornified envelope in the skin and interacts with various types of proteins in cultured cells; its level decreases dramatically during tumorigenic progression in human epithelial tissues. Despite these intriguing observations, the physiological roles of PPL, especially in noncutaneous tissues, are still largely unknown. Because we observed a marked fluctuation of PPL expression in mouse liver in association with the bile acid receptor farnesoid X receptor (FXR) and cholestasis, we sought to characterize the role of PPL in the liver and determine its contributions to the etiology and pathogenesis of cholestasis.
Post-diagnosis hemoglobin change associates with overall survival of multiple...Enrique Moreno Gonzalez
Anemia refers to low hemoglobin (Hb) level and is a risk factor of cancer patient survival. The National Comprehensive Cancer Network recently suggested that post-diagnosis Hb change, regardless of baseline Hb level, indicates the potential presence of anemia. However, there is no epidemiological study evaluating whether Hb change has direct prognostic values for cancer patients at the population level.
Cost-effectiveness of MRI for breast cancer screening in BRCA1/2 mutation car...Enrique Moreno Gonzalez
Women with mutations in BRCA1 or BRCA2 are at high risk of developing breast cancer and, in British Columbia, Canada, are offered screening with both magnetic resonance imaging (MRI) and mammography to facilitate early detection. MRI is more sensitive than mammography but is more costly and produces more false positive results. The purpose of this study was to calculate the cost-effectiveness of MRI screening for breast cancer in BRCA1/2 mutation carriers in a Canadian setting.
Impaired mitochondrial beta-oxidation in patients with chronic hepatitis C: r...Enrique Moreno Gonzalez
Hepatic steatosis is often seen in patients with chronic hepatitis C (CH-C). It is still unclear whether these patients have an impaired mitochondrial β-oxidation. In this study we assessed mitochondrial β-oxidation in CH-C patients by investigating ketogenesis during fasting.
Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on...Enrique Moreno Gonzalez
Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics.
The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-i...Enrique Moreno Gonzalez
Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT1R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT2R) is lost. Further, our previous data indicate that patients with high CysLT1R and low CysLT2R expression have a poor prognosis. In this study, we examined whether the balance between CysLT1R and CysLT2R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA).
Clinical features and outcome of cryptogenic hepatocellular carcinoma compare...Enrique Moreno Gonzalez
Cryptogenic hepatocellular carcinoma (HCC) is thought to arise due to non-alcoholic fatty liver disease (NAFLD). This study investigated the prevalence, clinical features, and outcomes of cryptogenic HCC and compared them with those of HCC related to hepatitis B virus infection (HBV-HCC), hepatitis C virus infection (HCV-HCC), and alcohol (ALCHCC) in Korea.
Fatty liver index correlates with non-alcoholic fatty liver disease, but not ...Enrique Moreno Gonzalez
Fatty liver index (FLI) was recently established to predict non-alcoholic fatty liver disease (NAFLD) in general population, which is known to be associated with coronary artery atherosclerotic disease (CAD).
This study aims to investigate whether FLI correlates with NAFLD and with newly diagnosed CAD in a special Chinese population who underwent coronary angiography.
Antibiotic exposure and the development of coeliac disease: a nationwide case...Enrique Moreno Gonzalez
The intestinal microbiota has been proposed to play a pathogenic role in coeliac disease (CD). Although antibiotics are common environmental factors with a profound impact on intestinal microbiota, data on antibiotic use as a risk factor for subsequent CD development are scarce.
Optimal schedule of Bacillus Calmette-Guerin for non-muscle-invasive bladder ...Enrique Moreno Gonzalez
To explore the necessity of maintenance, efficacy of low-dose and superiority of various combination therapies of Bacillus Calmette-Guérin (BCG) in treatment of superficial bladder cancer (BCa).
Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, hypoxia. It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on four different cell lines in conditions of normoxia, hypoxia and anoxia.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
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The Central Drugs Standard Control Organization (CDSCO) is India's national regulatory body for pharmaceuticals and medical devices. Operating under the Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, the CDSCO is responsible for approving new drugs, conducting clinical trials, setting standards for drugs, controlling the quality of imported drugs, and coordinating the activities of State Drug Control Organizations by providing expert advice.
Pharmacovigilance, on the other hand, is the science and activities related to the detection, assessment, understanding, and prevention of adverse effects or any other drug-related problems. The primary aim of pharmacovigilance is to ensure the safety and efficacy of medicines, thereby protecting public health.
In India, pharmacovigilance activities are monitored by the Pharmacovigilance Programme of India (PvPI), which works closely with CDSCO to collect, analyze, and act upon data regarding adverse drug reactions (ADRs). Together, they play a critical role in ensuring that the benefits of drugs outweigh their risks, maintaining high standards of patient safety, and promoting the rational use of medicines.
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Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
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Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
2. OSU-03012 sensitizes breast cancers to lapatinib-
induced cell killing: a role for Nck1 but not Nck2
N Winston West1
Email: norvell.west@richmond.edu
Aileen Garcia-Vargas2
Email: Garciavargam@vcu.edu
Charles E Chalfant2*,3,4
*
Corresponding author
Email: cechalfant@vcu.edu
Margaret A Park2*
*
Corresponding author
Email: mpark4@vcu.edu
1
Department of Biology, University of Richmond, 28 Westhampton Way,
Richmond, VA 23173, USA
2
Virginia Commonwealth University, Department of Biochemistry, Cell and
Molecular Biology, Sanger Hall, 1101 E. Marshall St, Richmond, VA 23298,
USA
3
Virginia Commonwealth University, Massey Cancer Center, 401 College Street,
Richmond, VA 23298, USA
4
Hunter Holmes McGuire VAMC, 1201 Broad Rock Blvd, Richmond, VA
23249, USA
Abstract
Background
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved
for the treatment of metastatic breast cancer. In this study, we examined mechanisms
associated with enhancing the activity of lapatinib via combination with other therapies.
Methods
In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells
were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1,
or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for
cytotoxicity/cytostaticity using clonogenic assays.
3. Results
Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule
derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects.
This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at
serine51
and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α
(Ser51
Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase
PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of
Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated
cells. Downregulation of Nck1 failed to “rescue” the ablation of the cytotoxic/cytostatic
effects by the phospho-mutant of eIF2-α (Ser51
Ala) demonstrating that Nck1 downregulation
is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and
OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α
dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.
Conclusions
These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination
therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this
complex is a novel target for the treatment of metastatic breast cancer.
Keywords
Breast cancer, Lapatinib, Combination therapy, Nck, eIF2-alpha
Background
Breast cancer is currently the second most common cause of death due to cancer among
women and leads to approximately 8,000 to 10,000 deaths per year [1]. Metastasis is the main
cause of breast cancer related deaths, and these metastases are only poorly controlled with
first generation therapies such as taxanes [2-4]. Both the ErbB2 and the ErbB1 receptors,
members of the epidermal growth factor receptor (EGFR) family, are upregulated in many
types of cancer, and overexpression of these proteins is associated with a greater likelihood of
metastasis. Hence, this receptor family is a current therapeutic target for the treatment of
metastatic breast cancer.
The epidermal growth factor receptor family comprises four members known as EGFR
(ErbB1), Her2 (ErbB2), ErbB3, and ErbB4. Homo- and hetero-dimerization of these tyrosine
kinase receptors occurs as a result of binding by various growth factors such as epidermal
growth factor (EGF), after which cytoplasmic tail tyrosine residues are phosphorylated [5,6].
Phosphorylation leads downstream to the activation of various signaling cascades such as the
extracellular-regulated kinase (ERK), and the Akt kinase cascades. These cascades lead to
propagation of both survival and death signals [7,8]. Recently, lapatinib (Tykerb, GSK), an
ErbB1/2 inhibitor, was approved for the treatment of metastatic breast cancer, as lapatinib is
implicated in better outcomes in patients with metastases. Unfortunately, outcomes are still
not ideal for patients with metastatic disease [9,10]. Thus therapies which enhance lapatinib-
induced cell killing are needed in the clinic.
4. One possibility for combination therapy with lapatinib is the small molecule inhibitor, OSU-
03012. This novel Celecoxib derivative induces death in cancer cells from multiple lineages
without inhibiting Cox-2 [11-14]. Previous analyses indicate that OSU-03012 induces cell
death partially via the activation of ER stress proteins including PKR-like ER kinase (PERK).
PERK is a direct kinase of the eukaryotic initation factor 2 (eIF2) and phosphorylates this
protein at the serine51
residue of the alpha subunit [15,16]. Phosphorylation of eIF2-α leads to
increased expression of the pro-apoptotic transcription factor CHOP as well as the expression
of HSP70 family chaperones. Our previous analyses demonstrated that OSU-03012 reduced
Grp78/BiP levels and increased HSP70 levels in a PERK-dependent fashion [11,12]. The
laboratory of Dr. Chen, in general agreement with our previous studies, has shown that
inhibition of ErbB1 in ErbB1-addicted NSCLC enhances the toxic effects of OSU-03012, and
that this is in part due to increased ER stress signaling and increased levels of DR5 [14]. The
laboratory of Dr. Paul Dent has also recently published that OSU-03012 and lapatinib
synergize in glioblastoma cell lines, although by a different mechanism than the one found in
this manuscript [17].
In the current studies, we assessed whether OSU-03012-induced killing of breast cancer cell
lines was enhanced by the addition of lapatinib. We show that a decrease in adaptor protein
Nck1, but not Nck2, [18,19] is necessary for cell killing in both ER positive and ER negative
breast cancer cell lines. Furthermore, we show that increased eIF2-α phosphorylation on
Serine51
induced by the combination of OSU-03012 and lapatinib is responsible for the
synergistic effects of these agents. Thus, the Nck1/eIF2 complex is identified in this study as
a novel target for the treatment of metastatic breast cancer.
Methods
Cell culture
The MDA-MB-231 cell line (purchased from American Type Culture Collection, ATCC) and
the BT474 cell line (ATCC) were maintained in RPMI (Invitrogen). ATCC published
standards are recognized by the American National Standards Institute (ANSI) and are
compatible with the requirements of the International Organization for Standardization (ISO).
Both cell lines were supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1%
Penicillin / Streptomycin (Invitrogen). All cell lines were maintained in a 95% air / 5% CO2
incubator at 37˚C. Cells were passaged once every 3-5 days (~90% confluence), and all
experiments were performed during the first 12 passages.
Plasmids and reagents
eIF2-α expression plasmids were constructed by Ron et. al. and purchased from Addgene
(plasmid numbers #21808 and 21807, [20]). GFP-tagged Nck1 and Nck2 plasmids were a
generous gift from Dr. L. Larose [18,19]. Antibodies to Nck1, phospho-eIF2-α (Serine51
),
total eIF2-α, ERK, phospho-ERK, PTEN, phospho-PTEN, PP1, phospho-PP1 and β-actin
were purchased from Cell Signaling Technologies. Nck2 antibodies were purchased from
Novus Biologicals. siRNA molecules against Nck1 and mutant siRNA molecules were
custom manufactured by Dharmacon. The sequence used was previously published by Dr. W.
Li and colleagues [21]. A mutant sequence containing 9 mutations was also manufactured as
a control to ensure specificity of knockdown. Sequences are as follows (single stranded,
sense): siNck1 5’ GGC CTT CAC TCA CTG GAA A 3’; Mutant Nck1 5’ CGC TTC CAC
5. TGC TGA GAG A 3’. Pre-designed and validated siRNA molecules to downregulate eIF2-α
and control scrambled siRNA molecules were purchased from Qiagen. siRNAs targeting
ATF6 and IRE-1 were generous gifts from the laboratory of Dr. Paul Dent.
Apoptosis assays
Cells were treated as indicated. 24 - 48 hrs later, cells were trypsinized, washed and stained
with Annexin V-PE and propidium iodide using the ApoScreen Annexin V Apoptosis Kit
(Southern Biotech) according to manufacturer’s instructions. Cells were detected using a BD
FACSCanto II and analyzed using the accompanying FACSDIVA software.
Transfection (plasmid)
Plasmid transfections were accomplished using the Effectene system (Qiagen) according to
manufacturer’s instructions. Briefly, plasmid DNA (1 µg) was incubated in the presence of
EC buffer and a 150:18 dilution of the Enhancer reagent for 10 minutes followed by the
addition of the Effectene reagent (at a 168:20 dilution). Plasmid samples were incubated for a
further 10 minutes then diluted to 1 mL with complete medium and added by single drops to
the sample. Cells were allowed to accumulate the recombinant proteins for 24-48 hours. All
steps excluding the incubation of DNA, EC buffer, Enhancer reagent and Effectene reagent
were undertaken in 10% FBS-containing medium.
Transfection (siRNA)
siRNA transfections were performed using the Dharmafect 1 reagent (Dharmacon) according
to manufacturer’s instructions. Briefly, siRNA molecules (25 nM final concentration) were
incubated in serum- and antibiotic-free medium. Concurrently, 5 µL Dharmafect 1 reagent
was incubated in serum- and antibiotic-free medium. Both tubes were incubated at room
temperature for 10 minutes then combined and incubated at room temperature for an
additional 20 minutes. siRNA was then added to cells one drop at a time. Cells were
incubated for at least 48 hours to achieve downregulation of the target mRNA.
Survival assays
Clonogenic assays were performed as previously described [22]. Briefly, cells were
transfected and treated as indicated in the figure legends. Cells were then plated onto 6-well
plates at a density of 200-400 cells / well and allowed to form colonies over the next 10-14
days. Colonies were stained using crystal violet stain, and cells that underwent ≥ 50
doublings were counted as a colony.
Western blotting
Cells were plated, cultured and treated as indicated. Cells were washed 2 times in PBS and
lysed using CelLytic (Sigma) lysis buffer supplemented 1:100 with protease and phosphatase
inhibitors (Cell Signaling) and by sonication. Protein concentration was assessed using Bio-
Rad protein assay reagent. Equal amounts (10-20 µg) of protein were subsequently
electrophoresed on 10-12% SDS polyacrylamide gels and electrophoretically transferred to
PVDF membranes (Bio-Rad). Membranes were blocked in PBS supplemented with 0.1%
6. TWEEN-20 and 5% dry milk and exposed to primary and secondary antibodies as indicated.
Membranes were developed using SuperSignal West reagents (Pierce).
Co-immunoprecipitation assays
Cells were treated as described in figure legends. Cells were then harvested using NP-40
buffer (20 mM Tris-HCl, pH 8.0; 137 mM NaCl; 2 mM EDTA; 2% NP-40; protease and
phosphatase inhibitor cocktail (added prior to use)). Lysate was pre-incubated with protein
A/G agarose beads (1 h, 4˚C with rotation). Concurrently, Protein A/G agarose beads (Pierce)
were incubated with antibodies raised against either total eIF2-α or total PP1 (Cell Signaling).
Beads were washed 3 times with NP-40 buffer and then added to cell lysates. Lysates + beads
were incubated at 4˚C for 4 – 16 h with rotation and washed 3 times in NP-40 buffer. Bound
proteins were released from the antibody-coated beads using 200 mM glycine, pH 3.0.
Electrophoresis and western blotting procedures were then performed as previously
described.
Isobologram analyses
Isobologram analyses were performed using the method of Chou and Talalay [23]. Briefly,
colony formation assays were performed using stepwise increasing concentrations of OSU-
03012 and lapatinib either singly or in combination (1 µM, 2 µM and 3 µM both in single and
in combination treatments). Analyses were then performed using the Calcusyn program
(Biosoft). Fraction affected (FA) was calculated and the combination index (CI) was then
used as a measure of synergy.
Statistics
All P values refer to paired student’s t-tests; differences with p≤0.05 were considered
significant. Analyses were performed using the Sigmaplot software.
Results and discussion
OSU-03012 and lapatinib synergize to induce cell death in both ER positive and ER negative
breast cancer cell lines. As stated previously, one possibility for combination therapy with
the FDA-approved drug lapatinib is the small molecule OSU-03012 as this novel Celecoxib
derivative induces cell death in cancer cells from multiple lineages [11-14]. In our initial
studies, cell death (via annexin V-PE) of MDA-MB-231 (ER negative, [24]) and BT474 (ER
positive, [24]) breast cancer cells was assessed after co-treatment with OSU-03012 and
lapatinib. Neither OSU-03012 nor lapatinib at 1 or 2 µM (well below the maximum tolerated
dose) induced significant increases in cell death when compared to control conditions (Figure
1A-C). However, treatment of BT474 cells with single agents at 3 µM resulted in decreases in
clonogenic capacity when compared to controls (Fig. 1A). Treatment with the combination at
all concentrations tested showed a greater than additive effect (See Table 1, Figure 1). This
effect was confirmed by repeating the experiment and demonstrating a decrease in the
survival of cells treated with the combination at 2 µM (see Figure 1D-E). Synergy was
confirmed by survival assays followed by isobologram analyses (Table 1, [23]). A
combination index (CI) value of less than 1 indicates synergistic effects, whereas a CI value
of 1 indicates an additive effect and a CI value of greater than 1 indicates antagonistic effects.
These data demonstrate that OSU-03012 and lapatinib act synergistically to induce cell death
7. in both ER positive and ER negative breast cancer cell lines and provided a rationale for
treatment of cell lines at 2 µM for the remainder of the studies.
Figure 1 OSU-03012 and Lapatinib act to Kill ER-positive and ER-negative Breast
Cancer Cells in Combination. A, D-E: ER-positive BT474 and ER negative MDA-MB-231
cell lines were treated with the indicated concentrations of OSU-03012 and lapatinib (A) or 2
µM lapatinib OSU-03012 and 2 µM lapatinib (D-E) for 48 h. Cells were then singly plated
onto 6-well plates to assay for clonogenic capacity. B-C: BT474 and MDA-MB-231 cells
were treated with 2 µM OSU-03012 (OSU) or 2 µM lapatinib (Lap) or the combination,
incubated for 48 h, then assayed using Annexin V/PI for cell death. All measurements are ±
SEM. * indicates a p < 0.05 when compared to the vehicle-treated condition.
Table 1 Isobologram analysis of MDA-MB-231 and BT474 cell lines indicates that the
drugs are synergistic in multiple breast cancer lines
Drug Conc Lap Drug Conc. OSU FA CI Value
1uM 1uM .40 .12
2uM 2uM .41 .24
3uM 3uM .31 .24
BT474
1uM 1uM .5 .35
2uM 2uM .41 .52
3uM 3uM .28 .48
Interestingly, OSU-03012 and lapatinib combination therapy was more effective against
MDA-MB-231 cells than BT474 cells. Therefore, our findings argue that targeting ER stress
proteins may increase the efficacy of traditional therapies specifically for metastatic breast
cancers [11-13] since the BT474 cell line is less invasive than the triple negative MDA-MB-
231 cell line [25,26]. Specifically, we found a greater decrease in cell viability and a lower CI
value for synergy between OSU-03012 and lapatinib in the triple negative cell line MDA-
MB-231 (harvested from the metastatic pleural ascites) than in ErbB2-amplified BT474 cell
line (harvested from a primary site). These findings provide support for the hypothesis that
OSU-03012 and lapatinib in combination may be more effective against metastatic breast
cancers than non-metastatic breast cancers. These results are also in line with recent studies
by Sanz-Pamplona et. al., which showed that upregulation of GRP94, an ER stress protein, is
an effective marker for brain metastases of breast cancers [27], and others [28], which
showed that other ER stress markers are upregulated during suspension conditions.
Our data demonstrating that MDA-MB-231 cells are more sensitive to the combination of
OSU-03012/lapatinib are also in general agreement with the findings in Figure 7B, that PP1
associates significantly less with eIF2-α after OSU/lapatinib treatment in MDA-MB-231 cells
than in BT474 cells. While PTEN, Raf, and Akt levels and mutation status appear to be
similar in both MDA-MB-231 and BT474 cells [29-31], BT474 cells express a constitutively
active form of PI3KCA (K111N), in addition to overexpressing ErbB2 [32]. It may be that
upregulation of the PI3K/Akt pathway represents a potential pathway of resistance for cell
lines treated with OSU-03012/lapatinib in combination. Therefore, inhibitors of the PI3K
pathway should be combined with OSU-03012/lapatinib in future studies.
Phosphorylation of eIF2-α at serine51
specifically induces cell death in response to OSU-
03012 and lapatinib via protein phosphatase-1. Previous analyses indicate that OSU-03012
8. induces cell death partially via the activation of ER stress proteins, including PKR-like ER
kinase (PERK, [14] see Fig. 2), and that the ER stress response is important in breast cancer
tumorigenesis [27,28]. We therefore determined whether downregulation of the three main
ER stress sensors (PERK, IRE-1α and ATF6) decreased cell death induced by OSU-03012
and lapatinib in combination. The involvement of PERK in lapatinib/OSU-03012-induced
cytotoxicity was confirmed in these studies. Other ER stress sensors did not protect against
lapatinib/OSU-03012-induced cytotoxicity/cytostaticity (ATF6), or had a small protective
effect (IRE-1α, see Fig. 2). We therefore chose to focus on PERK-mediated effects for the
remainder of these studies. PERK is a direct kinase of the eukaryotic initation factor 2 (eIF2),
phosphorylating this protein at the serine51
residue of the alpha subunit [15]. Thus, the
phosphorylation state of eIF2-α was assessed in these studies as an indicator of ER stress.
Surprisingly, treatment of breast cancer cells with OSU-03012 or lapatinib alone only
affected the phospho-state of eIF2-α on Ser51
in a minor fashion (Figure 3). Importantly, the
phosphorylation of this protein was increased significantly after co-treatment lapatinib and
OSU-03012.
Figure 2 ER stress via PERK activation may be responsible for lapatinib/OSU-03012-
induced cytotoxicity/cytostaticity. A-B: MDA-MB-231 cells, 24 h after plating, were
transfected with the indicated siRNA. After a 24 h incubation, cells were either plated singly
onto 6-well plates and allowed to attach overnight (A) or harvested for immunoblotting to
ensure knockdown (B). Cells in (A) were treated with vehicle or OSU-03012/lapatinib (48 h)
then media was replaced and colonies were allowed to develop over the next 10-14 d.
Colonies were counted using crystal violet stain and the number of colonies was graphed
(n=3, *=p<0.05).
Figure 3 Phosphorylation of eIF2-α indicates ER stress signaling in MDA-MB-231 and
BT474 cells after treatment with OSU-03012 and lapatinib. MDA-MB-231 cells and
BT474 cells (1 x 106
) were subjected to vehicle (DMSO, Ctr), OSU-03012 (2 µM), lapatinib
(2 µM) or the combination as indicated for 3 h. Cells were then lysed and protein extracts
(10-20 µg) were subjected to SDS PAGE followed by western immunoblotting for the
indicated proteins.
Since eIF2-α phosphorylation on Ser51
was upregulated by combination therapy (Figure 3),
the role of eIF2-α was examined in the synergistic killing of breast cancer cells. As shown in
Figure 4A and B, knockdown of eIF2-α completely ablated the decrease in survival induced
by OSU-03012 and lapatinib. Importantly, ectopic expression of the inactive Ser51
Ala
phospho-mutant attenuated cell death induced by the combination treatment in contrast to
ectopic expression of wild-type eIF2-α (Figure 4C and D). These data demonstrate that eIF2-
α phosphorylation on serine51
is a central event in the induction of cell death induced by
OSU-03012 and lapatinib.
Figure 4 The role of eIF2-α phosphorylation in cell death induced by OSU-03012 and
lapatinib. A-B: MDA-MB-231 (A) or BT474 (B) cells (5 x 105
) were transfected with the
indicated siRNA molecules and incubated for 48 h. Cells were then treated with either
vehicle (Veh) or the combination of OSU-03012 (2 µM) and lapatinib (2 µM) (combo) as
indicated and either subjected immunoassays (bottom panels) or plated for clonogenic
capacity (top graphs). Numbers indicated are percent control (e.g. Veh). C-D: MDA-MB-231
cells (5 x 105
) were transfected with a control vector (Vector), wild-type (WT) or the
Ser51
Ala mutant (S51A) eIF2-α plasmids. After a further 24 h cells were plated onto 6-well
plates to assay for clonogenic capacity (D) or subjected to immunoblotting as described in
9. Materials and Methods (C). Cells were treated with either vehicle (Veh, DMSO) or OSU-
03012 (2 µM) and lapatinib (2 µM) in combination (combo) for 24 h (D) or 3 h (C). Colonies
were allowed to develop for the next 10-14 days. * indicates a p ≤ 0.05 with respect to
vehicle-treated cells. E: MDA-MB-231 cells (1 x 106
) were plated and treated with the
indicated drugs (Vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 µM), lapatinib (Lap, 2 µM) or
the combination (Combo)). Three hours later, cells were harvested and subjected to
immunoblotting. Samples were probed with the indicated antibodies (see Materials and
Methods).
PTEN [33] and protein phosphatase 1 (PP1, [34]) are two phosphatases whose activities are
linked to eIF2-α phosphorylation. Thus, we assessed the activity of these phosphatases as
upstream determinants of OSU-03012/lapatinib-induced eIF2-α phosphorylation. First, the
phospho-status of PTEN was examined as an indicator of activation, but no increases were
observed for the phosphorylation of PTEN (Figure 4E). Instead, the phosphorylation pattern
was similar to the pattern of total PTEN expression. Hence, enhanced PTEN activity is
unlikely affecting OSU-03012- and lapatinib-induced cell death/reduced survival. In Figure
4E, we observed that the phosphorylation of the PP1 was significantly increased indicating a
decrease in the activity of PP1 (Figure 4E, [34]). Thus, with regards to upstream events
leading to eIF2-α activation, PP1, but not PTEN, is a likely candidate responsible for the
dephosphorylation of eIF2-α induced by OSU-03012/lapatinib in combination.
Taken together, the data in Figures 3 and 4 showed that OSU-03012/lapatinib in combination
upregulated ER stress-related pathways, and that downregulation of eIF2-α phosphorylation
at serine51
completely ablated cell death induced by OSU-03012/lapatinib and demonstrated
that PP1 was a likely candidate for eIF2-α dephosphorylation.
ER stress aggravators (ERSAs) are a relatively recent addition to our arsenal of therapeutic
agents for the treatment of cancer. There are multiple reports [27,28,35] that ER stress factors
are upregulated in many types of cancer suggesting that these pathways may be ones to which
cancers may become addicted and therefore represent good targets for treatment. OSU-03012
represents one ERSA which may be used to enhance ER stress pathways in cancer cells. This
may activate a response in which the cancer cell shifts from using ER stress signaling as a
survival mechanism to an apoptotic one. Our findings demonstrate that eIF2-α
phosphorylation is a major event in the cell death pathways induced during treatment with
OSU-03012/lapatinib. Furthermore, the question whether other molecules that induce ER
stress will also enhance lapatinib-induced cell killing should be pursued in light of these
studies.
Nck1, but not Nck2 is intrinsic to OSU-03012/lapatinib-induced cell death
PP1 has been found by Larose et al [18,19] in a complex containing both eIF2 and the protein
Nck1. Nck1 (or Nckα), an SH-only adaptor protein, was originally characterized as playing a
role in driving cell motility [36], a hallmark of metastatic cancer. Nck1 binds to eIF2-β,
preventing the phosphorylation of eIF2-α specifically on Serine51
, and dissociation of Nck1
leads to increased levels of eIF2-α phosphorylation. Thus, we examined the role of Nck1 in
the enhanced phosphorylation of eIF2-α on Serine51
. A robust, greater-than-additive decrease
in the levels of Nck1 was observed in combination-treated samples (See Figure 5A, B) in
contrast to cells treated with a single drug. Nck2 (also known as Nckβ) expression did not
follow the same pattern indicating a novel differential role for these two family members in
OSU-03012- and lapatinib-induced cell killing.
10. Figure 5 Upstream signaling responsible for eIF2-α phosphorylation: A role for Nck1.
A-B: MDA-MB-231 cells and BT474 cells were treated with vehicle (Ctr, DMSO), OSU-
03012 (OSU, 2 µM), lapatinib (Lap, 2 µM) or the combination (Combo) for 3 h, then
harvested for immunoblotting assays as described in Materials and Methods. Membranes
were probed with the indicated antibodies. C: MDA-MB-231 cells (5 x 105
) were transfected
with plasmids to express either GFP-Nck1 or GFP-Nck2 as described. After an additional 24
h, cells were treated with the combination of OSU-03012 and lapatinib as indicated for 24 h
(upper graphs) or 3 h (lower panels), and then either plated onto 6-well dishes and allowed to
form colonies (graphs represent percent control) or harvested for immunoblotting assays and
probed with the indicated antibodies. * indicates a p<0.05.
Next, we examined the role of Nck1 in the cell death and eIF2 Ser51
phosphorylation induced
by the combination of OSU-03012 and lapatinib. The decrease in both clonogenic capacity
and eIF2-α phosphorylation in MDA-MB-231 cells after OSU-03012 and lapatinib
combination treatment was “rescued” by the ectopic expression of Nck1 (see Figure 5C), but
not by ectopically expressing Nck2. Furthermore, Nck1, when co-expressed with wild-type
eIF2-α, ablates the increase in cell death induced by OSU-03012 and lapatinib indicating a
role in the same pathway for this protein (See Figures 6A and C). In contrast, ectopic co-
expression of the Ser51
Ala phospho-deficient mutant of eIF2-α with either Nck1 or Nck2
ablated all cell death induced OSU-03012 and lapatinib in combination (See Figures 6B and
D). Co-expression of Nck2 and wild-type eIF2-α did not affect the levels of cell death
indicating that this pathway is specific for Nck1.
Figure 6 Nck1, but not Nck2 expression ablates the increase in cell death induced by
OSU-03012 and lapatinib. A-D: MDA-MB-231 cells (A, B), or BT474 cells (C, D) (5 x
105
) were co-transfected with either wild-type eIF2-α and the two Nck isoforms (A, C), or the
S51
A phospho-mutant and the two Nck isoforms (B, D). Cells were allowed to incubate for 24
h to induce ectopic expression, and then treated with either vehicle (indicated with a -), or the
combination of OSU-03012 and lapatinib (2 µM each, indicated with a +). Cells were either
harvested after a 3 h treatment for western blotting as described in Materials and Methods
(bottom panels), or plated singly onto 6-well plates to assay for clonogenic capacity (upper
graphs). Cells were treated with OSU-03012 (2 µM) and lapatinib (2 µM) for 24 h, and then
allowed to form colonies for 10-14 days. Total colony counts were graphed. * denotes a p-
value of <0.05.
Finally, in agreement with our hypothesis that decreased Nck1 expression is upstream to the
increase in eIF2-α phosphorylation, we showed that downregulation of Nck1 was insufficient
to re-sensitize BT474 cells to the ablation of OSU-03012 and lapatinib-induced cell death
when the phospho-mutant of eIF2-α is ectopically expressed (Figure 7A). In addition, OSU-
03012/lapatinib in combination induces a decrease in the association of eIF2-α with PP1
(Figure 7B). Taken together, these data demonstrate that a major mechanism of cell death via
the combination of OSU-03012 and lapatinib is a decrease in Nck1 expression followed by
upregulation of eIF2-α phosphorylation, and thus ER stress-related cell death (Figure 7C).
Figure 7 Nck1 is upstream of eIF2-α phosphorylation in the cell death induced by the
combination of OSU-03012 and lapatinib. A: BT474 cells (5 x 105
) were co-transfected
with the Ser51
Ala eIF2-α mutant and GFP-Nck1. Cells were allowed to incubate for 24 h to
induce ectopic expression then treated with either vehicle (indicated with a -), or the
combination of OSU-03012 and lapatinib (2 µM each, indicated with a +). Cells were either
harvested for western blotting as described previously (lower panel), or plated into 6-well
11. plates to assay for clonogenic capacity as described previously (upper graph). Graphs
represent total colony number. * denotes a p-value of <0.05. B: BT474 (upper panel) and
MDA-MB-231 (lower panel) cells were treated with either vehicle (Veh) or OSU-03012 and
lapatinib in combination (combo). PP1 was immunoprecipitated and the resulting membranes
were immunoblotted with eIF2α and PP1. C: Our data indicate that Nck1 and PP1, which are
originally in a complex with eIF2 are dissociated after treatment with the combination of
OSU-03012 and lapatinib. This event frees eIF2-α to become phosphorylated by one of many
upstream kinases such as PERK, leading to ER stress and eventual cell death.
Larose and colleagues [18,19] found that Nck1 forms a complex with eIF2 and PP1.
Dissociation of this complex can lead to eIF2-α phosphorylation at serine51
and a decrease in
protein translation. eIF2-α may also be phosphorylated at serine51
by the ER resident kinase
PERK during ER stress. Since we show in Figure 2 that OSU-03012/lapatinib in combination
induces ER stress in part by PERK activation, we performed studies aimed at determining the
role of Nck1 in ER stress-induced cell death by OSU-03012 and lapatinib in combination.
Our studies showed that ectopic expression of Nck1 abolished the cell death induced by
OSU-03012/lapatinib. Furthermore, upregulation of Nck1 “rescues” the cell death induced by
wild-type eIF2-α overexpression. Thus, the studies reported here demonstrate that the
Nck1/eIF2 complex is a key point at which lapatinib and OSU-03012 act to synergistically
kill metastatic breast cancer cells, and generally support Larose’s findings that PP1 is
important in this complex.
In contrast to our findings implicating a PP1, Nck1 and eIF2-containing complex in the
cytotoxicity/cytostaticity induced by OSU-03012/lapatinib, the Dent laboratory has recently
published that lapatinib enhances OSU-03012-induced cell killing in glioblastoma models
and that this phenomenon occurs via an ErbB/Akt/PTEN pathway [17]. MDA-MB-231 and
BT474 cells as well as GBM6 and GBM12 (used in [17]) cell lines are all PTEN wild-type.
Therefore, cancer-type-specific pathways may be responsible for this apparent contradiction.
Our data suggest that further experiments may need to take these cancer-specific differences
into account when designing therapeutic regimens.
Recently, EGFR-mediated Nck1/Rap1 activation has been shown to upregulate metastasis in
a model of metastatic pancreatic carcinoma without affecting primary tumor growth [37].
These findings raise two intriguing possibilities: 1) Nck1 downregulation may be a singularly
efficacious inducer of cell death specifically for metastatic breast cancer cells, and 2) eIF2
may play a role in the metastatic process. We observe a small, but insignificant decrease in
the viability of BT474 cells (a non-invasive cell line, see Figure 7) after RNAi-mediated
inhibition of Nck1, which may be indicative that inhibition of Nck1 alone may induce cell
death in more invasive cell lines. In addition, we observe that Nck1 is downregulated only
with the combination treatment in MDA-MB-231 (a more invasive cell line) cells even
though eIF2-α phosphorylation is upregulated in samples treated with single drugs. eIF-4E,
the mRNA cap-binding protein essential for the initiation of translation, has been found to
contribute to malignancy by enabling translation of select mRNAs that encode proteins
involved in growth, angiogenesis, survival and malignancy [38]. Interestingly, ER stress
signaling and eIF2-α phosphorylation have been linked to drug resistance and survival in
occult dormant carcinoma cells [39]. However, eIF2-α has never before been characterized
specifically as a regulator of metastasis. Therefore, studies aimed at characterizing the
involvement of eIF2 in metastasis, both in vivo and in vitro, are a natural continuation of
these findings as are studies aimed at examining the potential of Nck1 inhibition as a therapy
specific for metastatic breast cancer.
12. Conclusions
Combination therapies are especially useful in the treatment of many cancers, in part due to
the ability of separate drugs to target multiple separate survival pathways upregulated in
many cancer lineages [40]. In these studies, we have used the concept of combination
therapies to delineate the interaction between OSU-03012 and lapatinib. We showed that
OSU-03012 and lapatinib synergized to induce cell death in both an ER positive and an ER
negative breast cancer cell line suggesting that this therapeutic model may be effective
against a variety of breast cancer phenotypes. We also demonstrated that eIF2-α
phosphorylation is a central event in the synergistic cytotoxicity/cytostaticity induced by the
combination therapy of OSU-03012 and lapatinib, and that this event is partially mediated by
the protein phosphatase PP1/Nck/eIF2 complex.
These studies describe a novel mechanism of cytotoxicity/cytostaticity via Nck1-mediated
eIF2-α phosphorylation for the combination of lapatinib and OSU-03012. We conclude that
OSU-03012 and lapatinib act synergistically to induce cell death via the downregulation of
Nck1/PP1 and the subsequent dissociation of this complex from eIF2-α. We also conclude
that this dissociation likely leads to a PP1-mediated enhancement of eIF2-α phosphorylation
at serine51
, a marker for ER stress and a central event in the induction of cell death by OSU-
03012/lapatinib. This work additionally identifies the Nck1/PP1/eIF2-α as a novel target for
inhibition for future therapies.
Abbreviations
ErbB, Avian erythroblastosis oncogene B; ER, Estrogen receptor; eIF, Eukaryotic initiation
factor; ERK, Extracellular-regulated kinase; PKR, Protein kinase R; PERK, PKR-like ER
kinase; PP1, Protein phosphatase-1; DR5, Death receptor 5; PTEN, Phosphatase and tensin
homolog; PBS, Phosphate buffered saline; FBS, Fetal bovine serum; PVDF, Polyvinylidene
fluoride; FA, Fraction affected; CI, Combination index.
Competing interests
The authors wish to declare that they have no competing interests.
Authors’ contributions
NWW and AGV collected clonogenic and apoptosis data, and AGV performed siRNA
experiments. MAP and CEC were involved in the experimental design and conception. MAP
performed western blotting, siRNA and plasmid transfection, co-immunoprecipitation and
some clonogenic assays. MAP analyzed the data and wrote the manuscript with editorial
input from CEC. All authors read and approved the final manuscript.
Acknowledgements
We would like to acknowledge the laboratory of Dr. Louise Larose for their generous
contribution of the GFPNck1 and GFPNck2 expression plasmids, and the laboratory of Dr.
Paul Dent for their generous contribution of siRNA to ATF6 and IRE1α. This work was
13. supported by grants from the Veteran’s Administration (Research Career Scientist Award to
CEC BX001792), and from the National Institutes of Health (CA154314 (CEC), NH1C06-
RR17393 (to Virginia Commonwealth University for renovation).
Services and products in support of the research project were generated by the VCU Massey
Cancer Center Flow Cytometry Shared Resource, supported, in part, with funding from NIH-
NCI Cancer Center Support Grant P30 CA016059. These funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
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