Human Mammary Carcinoma Cells Research Articles

TNOX, an alternative target to COX-2 to explain the anticancer activities of non-steroidal anti-inflammatory drugs (NSAIDS)

Our work has identified a cancer-specific, cell surface and growth-related quinol oxidase with both NADH oxidase and protein disulfide-thiol interchange activities, a member of the ECTO-NOX protein family designated tNOX. We provide evidence for tNOX as an alternative drug target to COX-2 to explain the anticancer activity of COX inhibitors. Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib all specifically inhibited tNOX activity of HeLa (human cervical carcinoma) and BT-20 (human mammary carcinoma) cells (IC(50) in the nanomolar range) without effect on ECTO-NOX activities of non-cancer MCF-10A mammary epithelial cells. With cancer cells, rofecoxib was less effective and two NSAIDS selective for COX-1 were without effect in inhibiting NOX activity. The IC(50) for inhibition of tNOX activity of HeLa cells and the IC(50) for inhibition of growth of HeLa cells in culture were closely correlated. The findings provide evidence for a new drug target to account for anticancer effects of NSAIDS that occur independent of COX-2.

A Formylated Hexapeptide Ligand Mimics the Ability of Wnt-5a to Impair Migration of Human Breast Epithelial Cells

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt-5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated beta-catenin or active JNK. The novel formyl-Met-Asp-Gly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.

The magnitude and time-dependence of the apoptotic response of normal and malignant cells subjected to ionizing radiation versus hyperthermia

Purpose: The purpose of the study was to examine the optimal time of exposure and dose of heat and ionizing radiation that results in the killing of human cancer cells in vitro via apoptosis vs. necrosis.Materials and methods: Human mammary carcinoma, colorectal carcinoma and normal bovine capillary endothelial (BCE) cell lines were subjected to 20 Gy ionizing radiation and 6, 12, 24, and 72 h later assessed for apoptosis using detection of apoptotic bodies and caspase assays. Necrosis was detected by loss of cells from the surface and lactate dehydrogenase (LDH) release. The colorectal carcinoma cells were subjected to hyperthermia using temperatures ranging from 39 – 44°C for 5, 15 or 45 min. exposures and at varying times post-treatment, apoptosis and necrosis were measured.Results: In response to ionizing radiation, none of the cells underwent necrosis and some cell types apoptosed 24 and 72 h posttreatment. The colorectal cancer cells exhibited a steady increase of apoptosis at 6, 12, and 24 h. When these cells were exposed to 40°C for 5 min, caspases increased within 6 h and a significant fraction (50%) of cells apoptosed. If the time of exposure to 40°C was increased to 15 or 45 min, 80% and 100% of the dying cells apoptosed, respectively. A temperature of 39°C did not cause cell death even after 45 min exposures. If heat was elevated to 42 or 44°C, increased necrosis was observed with a corresponding decrease in apoptosis.Conclusions: These studies reveal time and temperature dependent in vitro cell responses to ionizing radiation and water-bath hyperthermia.

The epidermal growth factor stimulates sphingosine kinase-1 expression and activity in the human mammary carcinoma cell line MCF7

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [ 3H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.

High intensity focused ultrasound-induced gene activation in sublethally injured tumor cells in vitro

Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-microl transfected cells (5 x 10(7) cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P- = 2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 degrees C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 degrees C to 13% at 70 degrees C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 degrees C to 41% at 70 degrees C. A similar trend was observed at 60 degrees C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 degrees C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.

Transient exposure of mammary tumors to PD184352 and UCN-01 causes tumor cell death in vivo and prolonged suppression of tumor re-growth

Previous studies from our group have demonstrated in vitro that UCN-01 (7-hydroxystaurosporine) and inhibitors of MEK1/2 interact to cause tumor cell death in a wide variety of malignant, but not in non-transformed, cell types. The present studies determined whether UCN-01 and MEK1/2 inhibitors interacted to cause tumor cell death in vivo. In vitro colony formation studies demonstrated that UCN-01 and the MEK1/2 inhibitor PD184352 interacted to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) with similar combination index values. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of ~100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), UCN-01 (0.1-0.2 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following ~30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to either PD184352 or UCN-01 did not significantly alter tumor growth rate or the mean tumor volume in vivo ~15-30 days after drug administration. In contrast, combined treatment with PD184352 and UCN-01 significantly reduced MDA-MB-231, and largely abolished MCF7 tumor growth. Tumor control values for both cell lines were 0.36. Tumor cells isolated ~30 days after combined drug exposure exhibited a significantly greater reduction in plating efficiency using ex vivo colony formation assays than tumor cells that were exposed to either drug individually. Reduced tumor growth correlated with profound tumor cell death within 5 days of combined drug exposure, which was also evident ~30 days after exposure. In addition, tumor cell death correlated with a reduction in the phosphorylation of ERK1/2 and the immuno-reactivity of Ki67 and of CD31. Collectively, these findings argue that UCN-01 and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.

Metabolic Activation-related CD147-CD98 Complex

Cell surface CD147 protein promotes production of matrix metalloproteinases and hyaluronan, associates with monocarboxylate transporters and integrins, and is involved in reproductive, neural, inflammatory, and tumor functions. Here we combined covalent cross-linking, mass spectrometric protein identification, and co-immunoprecipitation to show selective CD147 association with three major types of transporters (CD98 heavy chain (CD98hc)-L-type amino acid transporter, ASCT2, and monocarboxylate transporters) as well as a regulator of cell proliferation (epithelial cell adhesion molecule). In the assembly of these multicomponent complexes, CD147 and CD98hc play a central organizing role. RNA interference knock-down experiments established a strong connection between CD147 and CD98hc expression and a strong positive association of CD147 (and CD98hc) with cell proliferation. As the CD147-CD98hc complex and proliferation diminished, AMP-activated protein kinase (a cellular "fuel gauge") became activated, indicating a disturbance of cellular energy metabolism. Our data point to a CD147-CD98 cell surface supercomplex that plays a critical role in energy metabolism, likely by coordinating transport of lactate and amino acids. Furthermore we showed how covalent cross-linking, together with mass spectrometry, can be used to identify closely associated transmembrane proteins. This approach should also be applicable to many other types of transmembrane proteins besides those associated with CD98hc and CD147.

Combination of 5-deoxy-5-fluorouridine and tamoxifen show cell-type specific antagonistic and cooperative effects on cytotoxicity in human mammary carcinoma cells.

The effect of tamoxifen (Tam) and 5-deoxy-5-fluorouridine (5-DFUR) cotreatment on cytotoxicity was assessed in various cancer-derived cell lines. Each cell line was treated with a range of Tam and 5-DFUR concentrations alone and in combination. The cell lines we examined include MDA-MB-231 (ERalpha-/ERbeta+), T47D (ERalpha+/ERbeta+), and three MCF-7 (ERalpha+/ERbeta+) sublines. Cell growth was assessed by MTT assay at multiple time points for up to 9 days. Fluorescence-associated cell sorting (FACS) analysis was used to examine the effects of Tam and 5-DFUR cotreatment on cell cycle progression. Treatment with Tam or 5-DFUR, which is metabolized to 5-FU in target cells, produced an anti-proliferative effect that was dose- and time-dependent in all cell lines examined; variation in cell line sensitivity to each compound and cotreatment was apparent. When 5-DFUR and Tam treatment were combined, T47D, MDA-MB-231, and an MCF-7 subline demonstrated a cooperative effect at the lowest Tam concentration tested in 9-day cultures. The significance of treatment dosage and duration was obvious when 5-DFUR and Tam cotreatment was observed to be antagonistic in 3-day cultures of a second MCF-7 subline and T47D cells. Results from our studies show that there may be cytotoxic benefits in the treatment of cancer from combined therapy with Tam and 5-FU precursor drugs. Enhanced inhibition of proliferation was observed when Tam was cotreated at low concentrations in relatively long-term cultures. This has clinical relevance in that it suggests that patients undergoing chemo-endocrine therapy with orally administered 5-FU precursor drugs may benefit from lower treatment dosages relative to other adjuvant therapies.

Observation of Incipient Tumor Angiogenesis That Is Independent of Hypoxia and Hypoxia Inducible Factor-1 Activation

It is well established that hypoxia potently stimulates tumor angiogenesis by activating hypoxia inducible factor-1 (HIF-1)-induced proangiogenic factors, such as vascular endothelial growth factor. However, very little is known about the role of hypoxia in incipient angiogenesis in avascular tumors during their early stages of growth. To noninvasively investigate the functional significance of hypoxia and HIF-1 activation in incipient tumor angiogenesis, we genetically engineered HCT116 human colon carcinoma cells and 4T1 mouse mammary carcinoma cells with constitutively expressed red fluorescence protein as a tumor marker and green fluorescence protein (GFP) as a reporter for hypoxia and HIF-1 activation. The accuracy of GFP fluorescence in reporting hypoxia was confirmed by flow cytometry analysis and by immunohistochemical comparison with pimonidazole, a well-established hypoxia marker drug. Mouse dorsal skin-fold window chambers showed that incipient angiogenesis preceded a detectable level of hypoxia. The detectable levels of hypoxia were spatially and temporally related with more intensive secondary angiogenesis following the initial onset of new vessel formation. Selective killing of hypoxic cells by tirapazamine efficiently eliminated or delayed the detection of hypoxic cells, but it did not significantly delay the onset of incipient angiogenesis. These findings provide the first in vivo evidence that incipient tumor angiogenesis may not depend on hypoxia or HIF-1 activation. This is in contrast to the clear role of hypoxia in driving angiogenesis once initial tumor microvessel formation has occurred.

Electrogene transfer of an Epstein–Barr virus‐based plasmid replicon vector containing the diphtheria toxin A gene suppresses mammary carcinoma growth in SCID mice

Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.

Gene Expression Profiling to Identify Oncogenic Determinants of Autocrine Human Growth Hormone in Human Mammary Carcinoma

We have exploited a discrepancy in the oncogenic potential of autocrine and exogenous human growth hormone (hGH) in an attempt to identify molecules that could potentially be involved in oncogenic transformation of the human mammary epithelial cell. Microarray analysis of 19,000 human genes identified a subset of 305 genes in a human mammary carcinoma cell line that were remarkably different in their response to autocrine and exogenous hGH. Autocrine and exogenous hGH also regulated 167 common genes. Semiquantitative reverse transcription-PCR confirmed differential regulation of genes by either autocrine or exogenous hGH. Functional analysis of one of the identified autocrine hGH-regulated genes, TFF3, determined that its expression is sufficient to support anchorage-independent growth of human mammary carcinoma cells. Small interfering RNA-mediated knockdown of TFF3 concordantly abrogated anchorage-independent growth of mammary carcinoma cells and abrogated the ability of autocrine hGH to stimulate oncogenic transformation of immortalized human mammary epithelial cells. Further functional characterization of the identified subset of specifically autocrine hGH regulated genes will delineate additional novel oncogenes for the human mammary epithelial cell.

Review of: Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone

The human growth hormone (hGH) gene is expressed in the normal human mammary epithelial cell and its expression increases concomitant with the acquisition of proliferative lesions. Herein we demonstrate that autocrine production of hGH in human mammary carcinoma cells dramatically enhances anchorage-independent growth in a Janus kinase 2-dependent manner. Forced expression of the hGH gene in immortalized human mammary epithelial cells increased proliferation, decreased apoptosis, altered the cellular morphology and resulted in oncogenic transformation. Autocrine hGH was therefore sufficient to support anchorage-independent growth of immortalized human mammary epithelial cells and tumor formation in vivo. Moreover, autocrine hGH disrupted normal mammary acinar architecture with luminal filling and deregulated proliferation in three-dimensional epithelial cell culture. Autocrine hGH utilized homeobox A1 to govern the transcriptional program required for autocrine hGH-stimulated oncogenic transformation of human mammary epithelial cells, including transcriptional up-regulation of c-Myc, cyclin D1, and Bcl-2. Forced expression of a single orthotopically expressed wild-type gene is therefore sufficient for oncogenic transformation of the immortalized human mammary epithelial cell.

Potent reversal of multidrug resistance by ningalins and its use in drug combinations against human colon carcinoma xenograft in nude mice

To evaluate the pharmacological properties and the possible therapeutic applications of a series of synthetic marine natural product analogs, ningalins (N1-N6), in terms of cytotoxicity, MDR-reversing activity, and enhancement of drug combinations with antitumor agents in vitro and in vivo. XTT assays, [3H]azidopine binding to P-glycoprotein (Pgp), cellular accumulation and efflux of labeled drugs were carried out in vitro. Drug combinations using combination index, dose-reduction index, and isobologram were performed in vitro and enhancement of efficacy in drug combinations against human colon carcinoma HCT-116 xenografts were conducted with nude mice. N3 at sub-IC50 cytotoxic concentration (10 microM) was capable of enhancing vinblastine (VBL) cytotoxicity toward human leukemic CCRF-CEM cells about 50,000-fold as measured by the decrease of IC50 of VBL. For CCRF-CEM/VBL1000 (1,500-fold resistant to VBL and overexpressing Pgp), N3 and N5 enhanced VBL cytotoxicity as much as 6.2 million-fold and 210,000-fold, respectively. Moreover, N3 and N5 collaterally made CCRF-CEM/VBL1000 cells 4,000-fold and 130-fold, respectively, more susceptible to VBL than the parent CCRF-CEM cells. In human mammary carcinoma cells MX-1/paclitaxel which were 170-fold resistant to taxol and 38-fold resistant to VBL, N3 was capable of enhancing VBL effect as much as 6,000-fold. Combination therapy on murine P388/doxorubicin (DX) leukemia with DX+N3 or taxol+N3 achieved greater efficacy than the therapy with each drug alone. Impressively, nude mice, bearing human colon carcinoma HCT-116 cells, treated with a suboptimal dosage of taxol in combination with N3, N5 or N6 led to shrinkage of established tumor and achieved total tumor remission, while taxol alone had no tumor disappearance in this xenograft model. Furthermore, the enhancement of antitumor effect by ningalins, at least in parts, are due to inhibiting Pgp which was supported by the observation that the ningalins compete for [3H]azidopine binding to Pgp, increase the cellular accumulation of VBL or taxol, and inhibit drug efflux from the tumor cells. The profound enhancement of antitumor cytotoxicity of vinblastine and taxol in vitro by ningalins may have multiple mechanisms including the MDR-reversing effects. The mechanisms for collateral sensitivity by ningalins against sensitive (parent) cells are not yet clear. The marked enhancement of therapeutic effect of taxol by ningalins against xenograft tumors in nude mice suggests potential applications of therapeutic use of ningalins.

Curcumin Selectively Induces Apoptosis in Deregulated Cyclin D1-expressed Cells at G2 Phase of Cell Cycle in a p53-dependent Manner

Curcumin (diferuloylmethane) is known to induce apoptosis in tumor cells. In asynchronous cultures, with time-lapse video-micrography in combination with quantitative fluorescence microscopy, we have demonstrated that curcumin induces apoptosis at G(2) phase of cell cycle in deregulated cyclin D1-expressed mammary epithelial carcinoma cells, leaving its normal counterpart unaffected. In our search toward delineating the molecular mechanisms behind such differential activities of curcumin, we found that it selectively increases p53 expression at G(2) phase of carcinoma cells and releases cytochrome c from mitochondria, which is an essential requirement for apoptosis. Further experiments using p53-null as well as dominant-negative and wild-type p53-transfected cells have established that curcumin induces apoptosis in carcinoma cells via a p53-dependent pathway. On the other hand, curcumin reversibly inhibits normal mammary epithelial cell cycle progression by down-regulating cyclin D1 expression and blocking its association with Cdk4/Cdk6 as well as by inhibiting phosphorylation and inactivation of retinoblastoma protein. In addition, curcumin significantly up-regulates cell cycle inhibitory protein (p21Waf-1) in normal cells and arrests them in G(0) phase of cell cycle. Therefore, these cells escape from curcumin-induced apoptosis at G(2) phase. Interestingly, these processes remain unaffected by curcumin in carcinoma cells where cyclin D1 expression is high. Similarly, in ectopically overexpressed system, curcumin cannot down-regulate cyclin D1 and thus block cell cycle progression. Hence, these cells progress into G(2) phase and undergo apoptosis. These observations together suggest that curcumin may have a possible therapeutic potential in breast cancer patients.

P44/42 MAP kinase-dependent regulation of catalase by autocrine human growth hormone protects human mammary carcinoma cells from oxidative stress-induced apoptosis

Previous microarray expression analyses have indicated autocrine human growth hormone (hGH) regulation of genes involved in the oxidative stress response. Expression analysis of antioxidant enzymes revealed that autocrine hGH increased both the mRNA and protein levels of catalase, superoxide dismutase 1 (SOD1), glutathione peroxidase and glutamylcysteine synthetase but not that of SOD2. As a consequence, autocrine hGH increased the antioxidant capacity of mammary carcinoma cells and protected against oxidative stress-induced apoptosis. Catalase activity was increased by autocrine production of hGH in mammary carcinoma cells and a catalase inhibitor abrogated protection from oxidative stress afforded by autocrine hGH. Autocrine hGH transcriptionally regulated catalase gene expression in a p44/42 MAP kinase-dependent manner and inhibition of MEK concordantly abrogated the protective effect of autocrine hGH against oxidative stress-induced apoptosis. Given that increased cellular oxidative stress is a key effector mechanism of specific chemotherapeutic agents, we propose that antagonism of autocrine hGH will improve the efficacy of chemotherapeutic regimes utilized for human mammary carcinoma.

Liposome encapsulated gold nanoshells for NanoPhototherapy induced hyperthermia

Gold nanoshells have been extensively studied since their invention and their role in both in vitro and in vivo photothermal therapy has been explored and demonstrated. In this work, we investigate liposomal delivery of gold nanoshells and evaluate their effects for in vitro NanoPhototherapy induced hyperthermia in human mammary carcinoma cells. In addition, we compare the application of liposome encapsulated gold nanoshells and free standing gold nanoshells in NanoPhototherapy. NanoPhotoTherapy induced hyperthermia was performed using a 785 nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, we monitored apoptosis by detecting capase-9 activity.

Photothermal Treatment of Human Carcinoma Cells Using Liposome-Encapsulated Gold Nanoshells

We report the application of liposome-encapsulated gold nanoshells for in vitro photo-induced hyperthermia in human mammary carcinoma cells. In addition to evaluating their effects in vitro, we compared the application liposome-encapsulated gold nanoshells and free-standing gold nanoshells for NanoPhotoTherapy (NPT). NPT-induced hyperthermia was performed using a 785-nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, we monitored the effectiveness of NPT by detecting apoptosis via capase-9 activity.

Oncogenic Transformation of Human Mammary Epithelial Cells by Autocrine Human Growth Hormone

The human growth hormone (hGH) gene is expressed in the normal human mammary epithelial cell and its expression increases concomitant with the acquisition of proliferative lesions. Herein we demonstrate that autocrine production of hGH in human mammary carcinoma cells dramatically enhances anchorage-independent growth in a Janus kinase 2-dependent manner. Forced expression of the hGH gene in immortalized human mammary epithelial cells increased proliferation, decreased apoptosis, altered the cellular morphology and resulted in oncogenic transformation. Autocrine hGH was therefore sufficient to support anchorage-independent growth of immortalized human mammary epithelial cells and tumor formation in vivo. Moreover, autocrine hGH disrupted normal mammary acinar architecture with luminal filling and deregulated proliferation in three-dimensional epithelial cell culture. Autocrine hGH utilized homeobox A1 to govern the transcriptional program required for autocrine hGH-stimulated oncogenic transformation of human mammary epithelial cells, including transcriptional up-regulation of c-Myc, cyclin D1, and Bcl-2. Forced expression of a single orthotopically expressed wild-type gene is therefore sufficient for oncogenic transformation of the immortalized human mammary epithelial cell.

Src Family Kinase Inhibitors Block Amphiregulin-Mediated Autocrine ErbB Signaling in Normal Human Keratinocytes

c-Src potentiates proliferation, survival, and invasiveness in response to epidermal growth factor (EGF) in human mammary carcinoma cells. Tyrosine (Tyr) 845 of ErbB1 is phosphorylated by Src and has been implicated in control of malignant behavior. Although several lines of evidence also suggest important interactions of ErbB and Src family kinase signaling in normal epithelial cells, little is known about the mechanism of this interaction. Studying normal human keratinocytes (NHKs), here we demonstrate strong expression of the Src family kinases Src, Yes, and Fyn; Src family kinase-dependent stimulation of Tyr 845 by EGF; and potent inhibition of NHK proliferation and migration by two Src family kinase inhibitors PP1 and PD173952. EGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation occurred at much lower concentrations of EGF than required to phosphorylate Tyr 845. Moreover, the effect of Src family kinase inhibitors on EGF-stimulated ERK phosphorylation was transient, prompting a search for other targets of Src family kinase action. By enzyme-linked immunosorbent assay analysis, we found that three different Src family kinase inhibitors [6-(2,6-dichlorophenyl)-8-methyl-2-(4-morpholin-4-ylphenylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (PD173952), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (SU6656)] markedly inhibited elaboration of soluble amphiregulin by NHKs. The ErbB inhibitor PD158780 and the mitogen-activated protein kinase kinase inhibitor U0126 also markedly inhibited NHK proliferation, migration, and amphiregulin production. Together, these observations demonstrate that one or more Src family kinases act upstream as well as downstream of ErbB1 to promote amphiregulin-dependent autocrine stimulation of NHKs and suggest that autocrine NHK proliferation is more dependent upon ERK activation than upon Tyr 845 phosphorylation.