A Nomogram and Risk Classification System Predicting the Prognosis of Patients with De Novo Metastatic Breast Cancer Undergoing Immediate Breast Reconstruction: A Surveillance, Epidemiology, and End Results Population-Based Study.

Background The lifespan of patients diagnosed with de novo metastatic breast cancer (dnMBC) has been prolonged. Nonetheless, there remains substantial debate regarding immediate breast reconstruction (IBR) for this particular subgroup of patients. The aim of this study was to construct a nomogram predicting the breast cancer-specific survival (BCSS) of dnMBC patients who underwent IBR. Methods A total of 682 patients initially diagnosed with metastatic breast cancer (MBC) between 2010 and 2018 in the Surveillance, Epidemiology, and End Results (SEER) database were included in this study. All patients were randomly allocated into training and validation groups at a ratio of 7:3. Univariate Cox hazard regression, least absolute shrinkage and selection operator (LASSO), and best subset regression (BSR) were used for initial variable selection, followed by a backward stepwise multivariate Cox regression to identify prognostic factors and construct a nomogram. Following the validation of the nomogram with concordance indexes (C-index), receiver operating characteristic (ROC) curves, calibration curves, and decision curve analyses (DCAs), risk stratifications were established. Results Age, marital status, T stage, N stage, breast subtype, bone metastasis, brain metastasis, liver metastasis, lung metastasis, radiotherapy, and chemotherapy were independent prognostic factors for BCSS. The C-indexes were 0.707 [95% confidence interval (CI), 0.666-0.748] in the training group and 0.702 (95% CI, 0.639-0.765) in the validation group. In the training group, the AUCs for BCSS were 0.857 (95% CI, 0.770-0.943), 0.747 (95% CI, 0.689-0.804), and 0.700 (95% CI, 0.643-0.757) at 1 year, 3 years, and 5 years, respectively, while in the validation group, the AUCs were 0.840 (95% CI, 0.733-0.947), 0.763 (95% CI, 0.677-0.849), and 0.709 (95% CI, 0.623-0.795) for the same time points. The calibration curves for BCSS probability prediction demonstrated excellent consistency. The DCA curves exhibited strong discrimination power and yielded substantial net benefits. Conclusions The nomogram, constructed based on prognostic risk factors, has the ability to provide personalized predictions for BCSS in dnMBC patients undergoing IBR and serve as a valuable reference for clinical decision making.

Mst2 Overexpression Inhibits Thyroid Carcinoma Growth and Metastasis by Disrupting Mitochondrial Fitness and Endoplasmic Reticulum Homeostasis.

Although the incidence of thyroid carcinoma has increased over the past several decades, it has an excellent prognosis and overall 5-year survival, with a stable mortality rate, except in cases with advanced stages or rare malignant tumor types. Biomarkers have emerged as effective targets of molecular therapy against thyroid carcinoma due to their rapid and convenient detection; however, there has been little clinical application. Macrophage stimulating 2 (Mst2) is a proapoptotic protein with implications in carcinogenesis and metastasis. We found that Mst2 overexpression-induced endoplasmic reticulum (ER) stress in MDA-T32 thyroid carcinoma cells, accompanied by elevated caspase-12 activity, increased apoptotic rate, and reduced cell viability. In addition, Mst2 overexpression contributed to mitochondrial damage, as evidenced by increased mitochondrial oxidative stress and activated the mitochondrial apoptotic pathway. Inhibition of the JNK pathway abolished these effects. These results show Mst2 to be a novel tumor suppressor that induces mitochondrial dysfunction and ER stress via the JNK pathway. Thus, Mst2 could potentially serve as a biomarker for developing targeted therapy against thyroid carcinoma.

Association Between TP53 Mutation and Prognosis in Wilms Tumor: A Meta-Analysis.

BackgroundTP53 mutation has been suggested to have prognostic value for patients with Wilms tumor (WT), but the results are still controversial. Methods: Relevant studies published until August 1, 2019 were identified by searching PubMed, EMBASE and Cochrane Library. A random-effect model was performed to assess pooled data. Begg's and Egger's test were used to evaluate the potential publication bias. Sensitivity analysis was used to evaluate the stability of results. Results: A total of seven eligible articles were included. There was no significant difference in the risk of death among patients with WT with different TP53 mutation status (odds ratio [OR] = 3.09, 95% confidence interval[CI]: 0.81-11.84). Combined hazard ratio (HR) suggested that TP53 mutation had an unfavorable impact on overall survival (OS) (HR = 4.17, 95% CI: 1.97-6.36) and disease-free survival (DFS) (HR = 2.23, 95% CI: 1.29-3.17) in WT. Conclusions: This meta-analysis demonstrates that TP53 mutations are associated with poorer prognosis in WT.

FDA Approval Summary: Calaspargase Pegol-mknl For Treatment of Acute Lymphoblastic Leukemia in Children and Young Adults.

On December 20, 2018, the Food and Drug Administration approved calaspargase pegol-mknl (CALASP), an asparagine-specific enzyme, as a component of a multi-agent chemotherapeutic regimen for acute lymphoblastic leukemia (ALL) in pediatric and young adult patients age 1 month to 21 years. Efficacy was determined on the basis of achievement and maintenance of steady-state nadir serum asparaginase activity (NSAA) above 0.1 U/mL when using CALASP, 2,500 U/m2 intravenously, every 3 weeks. In a randomized comparison to pegaspargase (PEGASP) every 2 weeks, treatment with CALASP every 3 weeks had a similar safety profile and no substantial impairment in event-free survival. The pharmacokinetics of CALASP were studied when administered in combination with multiagent chemotherapy in 124 patients with B-cell ALL in Study AALL07P4 and Study DFCI 11-001. The results showed that 123 [99%, 95% confidence interval (CI), 96%-100%] of the 124 patients maintained NSAA >0.1 U/mL at weeks 6, 12, 18, 24, and 30 of post-induction phase. Maintaining adequate NSAA levels is critical to successful treatment of ALL. Herein, we describe the FDA review and approval of CALASP.See related commentary by Lew, p. 325.

Downregulation of Long Noncoding RNA HOTAIR and EZH2 Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Human Breast Cancer Cells.

BACKGROUND: The long noncoding RNA HOTAIR (HOX transcript antisense intergenic RNA) has been reported to be a biomarker for various malignant tumors; however, its involvement in breast cancer is not fully understood. The aim of this study was to investigate the effects involved with long noncoding RNA HOTAIR and EZH2 (enhancer of zeste homologue 2) on the processes of proliferation, invasion, migration, and apoptosis of breast cancer cells. MATERIALS AND METHODS: The expressions of HOTAIR and EZH2 in both normal human mammary epithelial cell (HBL-100) and breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR-3) were detected by means of reverse transcription-quantitative polymerase chain reaction. The MCF-7 cells that exhibited the highest HOTAIR expressions were selected for further studies and divided into the control, negative control, and small interfering RNA-HOTAIR groups. The proliferation, invasion, migration, and apoptosis of breast cancer cells were evaluated by MTT assay, Scratch test, Transwell assay, and flow cytometry, respectively. The combination of HOTAIR with EZH2 and PTEN was predicted by bioinformation, with a dual-luciferase reporter gene assay providing further verification. RESULTS: Initially, lower expressions of HOTAIR and EZH2 in the normal human mammary epithelial cells, while higher expressions in the breast cancer cells of MCF-7, MDA-MB-231, and SKBR-3 were detected. In addition, the downregulation of HOTAIR or silencing of EZH2 was revealed to repress the proliferation, invasion, and migration, while acting to promote the apoptosis of the breast cancer cells. Furthermore, HOTAIR could bind specifically to EZH2 and PTEN, highlighting the capability of HOTAIR to inhibit the expression of PTEN by recruiting EZH2 in breast cancer, while the TCGA database demonstrated the expressions of PTEN were lower in breast cancer cells. CONCLUSIONS: The study suggests the higher expressions of HOTAIR and EZH2 among three breast cancer cells. Furthermore, the downregulation of HOTAIR or silencing of EZH2 was noted to inhibit the proliferation, invasion, and migration of breast cancer cells, while promoting their apoptosis.

Celastrol inhibits colorectal cancer cell proliferation and migration through suppression of MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Colorectal cancer (CRC) is one of the most frequent malignant tumors. Signaling by the PI3K/AKT pathway is crucial for CRC development and progression, including proliferation and migration. Celastrol has an anticancer effect, but its mechanism needs to be determined. Here, we showed that celastrol suppressed CRC cell proliferation and migration. Celastrol treatment also decreased the PI3K/AKT pathway components, and MMP3 and MMP7 expression levels. In addition, knockdown of AKT, not mTOR, inhibited MMP3 and MMP7 expression levels and AKT silencing promoted the celastrol-induced effects on CRC cell proliferation and migration. Taken together, these findings indicated that the celastrol-induced antitumor effects were mediated through MMP3 and MMP7 by the PI3K/AKT signaling pathway.

The use of a stably expressed FRET biosensor for determining the potency of cancer drugs.

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.

H-Ras regulation of TRAIL death receptor mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.

[Development of a method for measuring dissolved reactive phosphorus (DRP) and dissolved ferrous iron in large batch in pore water samples of sediments with micro-volumes].

A method was established to measure the concentrations of dissolved reactive phosphate (DRP) and dissolved ferrous iron (Fe) in micro-volume solution samples through colorimetric determination in large batch using a 384-well Microplate Spectrophotometer. Concentrations of DRP and dissolved Fe were determined by the molybdenum blue and phenanthroline colorimetric methods, respectively. The results showed that the sample consumption used for each parameter was between 20 and 50 microL after dilution, and the detection limits for DRP and dissolved Fe were 0.006 mg x L(-1) and 0.010 mg x L(-1) respectively, while the analytical precision varied between 1% and 5%. The established method was applied to measure DRP and dissolved Fe in pore waters of sediment profiles in Lake Taihu, which were collected by a high-resolution Peeper (HR-Peeper) device with a vertical resolution of 2 mm. The results showed a simultaneous increase of DRP and dissolved Fe in their concentrations with the depth of two sediment profiles investigated.

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog, EB 1089.

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D3 (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D3 failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D3 or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D3 and EB 1089 in NSCLC.

Stilbene 5c, a microtubule poison with vascular disrupting properties that induces multiple modes of growth arrest and cell death.

The stilbene derivative, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding site in tubulin. The current studies were designed to investigate the effectiveness of stilbene 5c against the HCT-116 human colon cancer cell line and B16/F10 melanoma cells as well as human endothelial cell tube formation and tumor perfusion. Stilbene 5c produced a time-dependent decrease in cell viability in both cell lines and the capacity of the cells to proliferate was not restored upon removal of the drug. Treatment with stilbene 5c also promoted both senescence and autophagy in both cell lines. TUNEL and annexin 5 staining indicated that apoptosis also occurs in stilbene 5c-treated HCT-116 cells, but not in B16/F10 melanoma cells. DAPI staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells) indicative of mitotic catastrophe in HCT-116 cells but not in the B16/F10 melanoma cells. p53-null HCT-116 cells demonstrated a similar growth arrest/cell death response to stilbene as p53-wild type HCT-116 cells. Stilbene 5c also completely inhibited human endothelial cell tube formation on Matrigel, consistent with potential anti-angiogenic actions. Using a new method developed for monitoring the pharmacodynamic effects of stilbene 5c in vivo, we found that a single injection of stilbene 5c reduced tumor perfusion by 65% at 4h, returning to baseline by 24h, while subsequent daily injections of stilbene 5c produced progressively larger reductions and smaller rebounds. This work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of growth arrest and cell death coupled with anti-angiogenic and antivascular actions.

Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

Autophagy, cell death and sustained senescence arrest in B16/F10 melanoma cells and HCT-116 colon carcinoma cells in response to the novel microtubule poison, JG-03-14.

PURPOSE: Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line. METHODS: Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on ß-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays. RESULTS: Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy. CONCLUSIONS: Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.

The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep?

Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.

Mislocalization of death receptors correlates with cellular resistance to their cognate ligands in human breast cancer cells.

Multiple clinical trials are ongoing to evaluate the potential antitumor activity of human TNF variants, Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and its agonistic antibodies. These drug products act through the death receptors (DRs) TNF receptor 1 (TNFR1), Fas/CD95, DR4 (TRAIL-R1) and/or DR5 (TRAIL-R2), respectively. Therefore, characterization of the level and localization of DR expression in cancer cells is important for DR-targeted therapy. In this study, we examined the subcellular distribution of the four DRs in a panel of 10 human breast cancer cell lines by western blots and flow cytometry and 50 human breast tumors by immunohistochemistry. Despite their total protein expressions, the DRs were found to be absent on the surface of some cell lines. Consistent with this result, all four DRs were found to be mostly expressed in the cytoplasm and/or the nucleus of primary breast tumors (n=50). We further determined the growth inhibition activity (GI50) of the death ligands, recombinant human TNFα, FasL and TRAIL, and found a correlation with the subcellular localization of the corresponding DRs. These results demonstrate an aberrant expression of the death receptors in breast cancer cells, and suggest that the lack of surface DRs appears to be predictive of tumor resistance to DR-targeted therapies.

Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3.

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.

A switch between cytoprotective and cytotoxic autophagy in the radiosensitization of breast tumor cells by chloroquine and vitamin D.

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an "autophagic switch" from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3-radiation combination.

Expression of P-450(c11beta) in adrenal aldosterone-producing adenomas and nodular hyperplasia tissues.

BACKGROUND: Our previous study suggests that decreased P-450(c17alpha) expression correlated with the overproduction of aldosterone in APA and nodular hyperplasia in patients with primary aldosteronism. This study was performed to further investigate if P-450(c11beta) contributes to the overproduction of aldosterone in APA and nodular hyperplasia tissues. METHODS: Total RNA and protein were extracted from 7 cases of APA tissue, 3 nodular hyperplasia tissues, 7 normal adrenal glands. P-450(c11beta) mRNA was examined by dot blot and confirmed by Northern blot analysis and by realtime PCR. Protein expression level of P-450(c11beta) was also investigated by immunohistochemical staining and confirmed by Western blot. RESULTS: The relative expression level of P-450(c11beta) mRNA to beta-actin in APA, nodular hyperplasia and the normal adrenal gland group are 47 +/- 22%, 55 +/- 13%, 64 +/- 16% respectively by dot blot and are 94 +/- 18%, 101 +/- 20%, 112 +/- 62% respectively by Northern blot. These results are further confirmed by realtime PCR. This result was also supported by the relative protein expression level of P-450(c11beta) to beta-actin which are 118 +/- 15%, 107 +/- 32%, 108 +/- 22% respectively evaluated by Western blot. There was no significant difference in protein expression level of P-450(c11beta) among the normal adrenal gland tissues, APA and adrenal nodular hyperplasia tissue, either (P > 0.05). CONCLUSIONS: These results suggest that P-450(c11beta) is not a key contributor to the overproduction of aldosterone in APA and nodular hyperplasia and can not be considered as a potential marker to differentiate between them in patients with primary aldosteronism.

Influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to chemotherapy in breast tumor cells.

Studies were performed to determine the influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to adriamycin (doxorubicin) in four human breast tumor cell lines and one murine breast tumor line. Sildenafil did not interfere with the effectiveness of adriamycin in any of the cell lines tested. Sildenafil also failed to protect MDA-MB231 cells against the cytotoxicity of cisplatin, taxol or camptothecin. Sildenafil enhanced sensitivity to adriamycin markedly in the p53 mutant MDA-MB231 and p53 null MCF-7/E6 cells and moderately in the MCF-7/caspase 3 and 4T1 cell lines. In the MDA-MB231 cells, sildenafil increased the extent of DNA damage induced by adriamycin as well as the extent of apoptotic cell death. Sildenafil did not influence sensitivity to adriamycin in bone marrow cells or macrophages. In an immunocompetent model of breast cancer (4T1 mammary carcinoma in Balb/c mice), sildenafil did not attenuate the antitumor effects of adriamycin; furthermore, the combination of sildenafil with adriamycin was no more toxic to the animals than adriamycin alone. Given that sildenafil has been shown to have the potential to protect the heart against the toxicity of adriamycin, these studies suggest that the inclusion of sildenafil with conventional chemotherapeutic protocols involving adriamycin (and possibly cisplatin, camptothecin and/or paclitaxel) should not compromise the antitumor effectiveness of these drugs nor enhance their toxicity to the patient.