ABSTRACT
BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.
Subject(s)
Breast Neoplasms , Lung Neoplasms , Humans , Female , Phosphatidylinositol 3-Kinases , Cell Line, Tumor , Breast Neoplasms/pathology , Apoptosis , HSP40 Heat-Shock Proteins/pharmacology , HSP40 Heat-Shock Proteins/therapeutic use , Forkhead Box Protein O1/pharmacology , DNA Modification Methylases/pharmacology , DNA Modification Methylases/therapeutic use , Tumor Suppressor Proteins/pharmacology , Tumor Suppressor Proteins/therapeutic use , DNA Repair Enzymes/pharmacology , DNA Repair Enzymes/therapeutic useABSTRACT
The objective of this study was to validate the results from our published work and to test the robustness of our unique malignancy index as a (non-invasive) predictor of prostate cancer in fresh blood samples obtained from patients diagnosed with prostate cancer (PCa), benign prostatic hyperplasia (BPH), and healthy volunteers (Controls). The malignancy index was obtained by dividing the product of three biomarker values, [urokinase plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1), and prostate-specific antigen (PSA)], by the age of the patient/healthy volunteer, using enzyme-linked immunosorbent (ELISA) assay methodology. The results confirmed earlier findings that the malignancy index discriminates prostate cancer from non-prostate cancer. The index significantly separated the PCa group from the Control group with values of 0.0701 (n=54) and 0.0007 (n=47), respectively, by a factor of 100. The malignancy index of the small BPH cohort was found to be 0.0016 (n=20), differing by a factor of 44 from the Control group. When data from the earlier study and the current study data were collectively analyzed, the index again significantly separated the PCa group from the Control group by a factor of 15, with values of 0.0624 (n=125) and 0.0042 (n=110), respectively. However, the same could not be said of the BPH data since the sample size (n=20) was well below par, for comparison. In the initial blood study, the PCa group was significantly separated from the Control group by a factor of 8.5. The data presented here concur with findings in needle biopsies and transurethral resection tissue, reported elsewhere (Bohm et al., 2013; Akudugu et al., 2015). At this preliminary stage, the malignancy index has potential and merit as a prostate cancer biomarker.
Subject(s)
Biomarkers, Tumor/blood , Prostatic Hyperplasia/diagnosis , Prostatic Neoplasms/diagnosis , Age Factors , Aged , Aged, 80 and over , Case-Control Studies , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay , Healthy Volunteers , Humans , Kallikreins/blood , Male , Membrane Proteins/blood , Middle Aged , Plasminogen Activator Inhibitor 1/blood , Predictive Value of Tests , Prostate/pathology , Prostate-Specific Antigen/blood , Prostatic Hyperplasia/blood , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathologyABSTRACT
No unambiguous role of the involvement of uroplasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) in prostate cancer has emerged, with current evidence suggesting that neither biomarker is likely of significant clinical value, save as an overall contributor. In this study, we attempt to discriminate prostate cancer from non-cancer in a cohort of plasma samples, using the Imubind ELISA assay. In this cohort, PAI-1 levels are higher in prostate cancer patients than healthy donors; uPA levels are higher in healthy donors than prostate cancer patients; and the uPA/PAI-1 ratio is higher in healthy donors than in prostate cancer patients. To date, and across three prostate sample types, i.e. transurethral resection tissue, needle biopsies, and blood plasma, data have been disparate. Given the inconsistency, a malignancy index was created by dividing the product of three biomarkers [uPA, PAI-1, and prostate specific antigen (PSA)] by the age of the patient/donor. The malignancy index clearly distinguishes prostate disease from non-disease in peripheral blood plasma samples (P=0.0127), concurring with findings in core needle biopsies and transurethral resection tissue, reported elsewhere. This is an important finding given the gravity of prostate cancer and the legions of over-diagnosed and over-treated men worldwide.
Subject(s)
Biomarkers, Tumor/blood , Neoplasm Proteins/blood , Plasminogen Activator Inhibitor 1/blood , Prostatic Neoplasms/blood , Urokinase-Type Plasminogen Activator/blood , Adult , Aged , Humans , Male , Middle Aged , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/pathologyABSTRACT
Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Receptor, ErbB-2/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Aniline Compounds/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Breast Neoplasms , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Humans , Imidazoles/pharmacology , Oxazoles/pharmacology , Quinolines/pharmacology , Sulfonamides/pharmacology , Triazoles/pharmacologyABSTRACT
Adrenal C19 steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11ß-hydroxyandrostenedione (11OHA4) and 11ß-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19 steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19 steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05µM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11ß-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03µM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19 metabolites at significantly higher levels than the C19 steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11ß-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.
Subject(s)
Adrenal Glands/metabolism , Androstenedione/analogs & derivatives , Prostatic Neoplasms/metabolism , Testosterone/analogs & derivatives , Aged , Androgens/metabolism , Androstenedione/metabolism , Cell Line, Tumor , Dihydrotestosterone/metabolism , Disease Progression , Gene Expression Regulation, Neoplastic , Glucuronic Acid/chemistry , Humans , Hydroxytestosterones/metabolism , Male , Steroids/chemistry , Steroids/metabolism , Tandem Mass Spectrometry , Testosterone/metabolismABSTRACT
Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5 Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6 h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors.
Subject(s)
Histones/metabolism , Lymphocytes/radiation effects , Radiation Monitoring/methods , X-Rays , Adult , Dose-Response Relationship, Radiation , Female , Flow Cytometry , High-Throughput Screening Assays , Humans , Lymphocytes/metabolism , Male , Middle Aged , Radiation Dosage , Young AdultABSTRACT
PURPOSE: The mode by which the xanthine derivative, pentoxifylline, induces a radiosensitizing effect in cell cultures is a key and controversial radiobiological issue and requires further elucidation. MATERIALS AND METHODS: Six human glioblastoma cell lines were tested for the effect of pentoxifylline treatment at maximum G2/M block on the basis of cell survival, mitotic activity, and micronucleus formation after exposure to gamma radiation. Cell survival was measured by the colony-forming assay. Micronucleus formation (an indicator of DNA damage) and the proportion of binucleated cells (a representation of mitotic activity) were determined using the cytokinesis-block assay. RESULTS: Remarkably, exposure to a single dose of 4 Gy produced strong G2/M blocks in both p53 mutant and wild-type cells. Addition of pentoxifylline at the peak of radiation-induced G2/M blocks resulted in a p53-independent reduction in cell survival in all cell lines. This radiosensitization was strongly correlated with the magnitude of the radiation-induced G2/M block. The changes observed in mitotic activity and micronucleus yield were also p53-independent. CONCLUSIONS: These results are at variance with the view that pentoxifylline preferentially sensitizes p53 mutant cells, and that sensitization occurs only when cells are irradiated in the presence of the drug. The data suggest that the effectiveness of pentoxifylline as radiosensitizer depends on the proportion of cells that are arrested in the G2/M phase transition following exposure to ionizing radiation. These findings can assist in the identification of cancers that may benefit from therapies using G2/M checkpoint abrogators.
Subject(s)
Cell Survival/radiation effects , Glioblastoma/pathology , Glioblastoma/physiopathology , Pentoxifylline/administration & dosage , Tumor Suppressor Protein p53/metabolism , Cell Line, Tumor , Humans , Radiation Tolerance/drug effects , Radiation Tolerance/radiation effects , Radiation-Sensitizing Agents/administration & dosageABSTRACT
The administration of cancer chemotherapeutic agents results in an increase in the apoptotic cells in the tumor: therefore, it has been assumed that anticancer drugs exhibit their cytotoxic effects via apoptotic signaling pathways. Characteristics that confer sensitivity to drug-induced apoptosis are, a functional p53 protein and expression of the apoptosis-promoting protein, bax. The role of p53 and bax/bcl-2 in drug-induced apoptosis was assessed in six prostate cell lines, 1532T, 1535T, 1542T, 1542N, BPH-1 and LNCaP using TD(50) concentrations of etoposide, vinblastine and estramustine. Cell death was monitored morphologically by fluorescent microscopy, and by flow cytometry (Annexin-V assay). Apoptotic morphology was rather low and ranged from 0.1% to 12.1%, 3.0% to 6.0% and 0.1% to 8.5% for etoposide, estramustine and vinblastine, respectively. Annexin-V binding and flow cytometry indicated apoptotic propensities of 0% to 4%, 0% to 3% and 0% to 5%, respectively. The percentage of cells responding to drug-induced apoptosis was, on average, higher in the tumor cell lines than in the normal cell lines, but showed no correlation with p53 status. The percentage of cells showing necrosis, assessed by Annexin binding and Propidium Iodide permeability in aqueous medium, tended to be much higher, and was found to be at the level of 5% to 30%. Immunoblotting demonstrated that bax and bcl-2 proteins were expressed at a basal level in all cell lines, but did not increase after exposure to TD(50) doses of the three drugs. The ratio of bax and bcl-2, measured by laser scanning densitometry, was not altered by the drug-induced DNA damage. The results suggest that apoptosis is not a major mechanism of drug-induced cell death in prostate cell lines and appears to be independent of p53 status and bax/bcl-2 expression.
Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Estramustine/pharmacology , Genes, bcl-2 , Genes, p53 , Prostatic Hyperplasia/genetics , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Vinblastine/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , DNA Damage , Dose-Response Relationship, Drug , Gene Expression Profiling , Humans , Male , Necrosis , Tumor Cells, CulturedABSTRACT
The relationship between radiosensitivity and DNA repair was investigated in six human prostate cell lines, 1542-NPTX, BPH-1, 1542-CP(3)TX, 1532-CP(2)TX, 1535-CP(1)TX and LNCaP. Except for LNCaP, these cell lines are new and were derived from primary prostate tumours and normal non-tumourigenic prostate tissue. Cell survival was assessed by clonogenic assay. DNA damage was determined in non-synchronised cells by constant-field gel electrophoresis, and expressed as the fraction of DNA released. For initial damage, cells were embedded in agarose and irradiated on ice with 0-100 Gy (60)Co gamma-irradiation. Residual DNA damage was measured after 2 h and 20 h of repair. Radiosensitivity, given as the mean inactivation dose, was found to vary between 1.62 and 2.77 Gy. We found that radiosensitivity significantly correlates with the 2 h DNA repair component, giving a correlation coefficient of 0.92 ( P=0.009). In the cell lines examined here the 2 h repair component emerges as an indicator of radiosensitivity.
Subject(s)
DNA Repair , DNA/radiation effects , Prostate/cytology , Radiation Tolerance , Cell Line, Tumor/cytology , Cell Line, Tumor/radiation effects , Cell Survival/radiation effects , Dose-Response Relationship, Radiation , Humans , Male , Prostatic Hyperplasia , Prostatic NeoplasmsABSTRACT
Chemotherapeutic drug resistance remains a significant obstacle in the control of prostate cancer. The influence of p53 and androgen status on the drug response of new cell lines from normal, benign and primary tumour epithelium was investigated. The prostate cell lines 1542-NPTX, BPH-1, 1542-CP(3)TX, 1532-CP(2)TX, 1535-CP(1)TX and LNCaP were exposed to TD(50) doses of etoposide, vinblastine and estramustine for a period of 24 h and re-incubated for a further 4 days before measuring the cell viability by crystal violet vital dye staining assay. The virus-transformed cell lines were found to be approximately ten times more sensitive to etoposide and vinblastine than the non virus-transformed LNCaP cell line. Estramustine proved to be the least toxic drug. The LNCaP cell line emerged as DHT-sensitive against nanomolar concentrations of 5alpha-dihydrotestosterone in charcoal-stripped growth medium. The virus-transformed cell lines were DHT-insensitive. Induction of p21 by (60)Co gamma-irradiation was used to assess the functionality of the p53 gene. p21 induction in the LNCaP cell line reached a peak 7.5 h post-irradiation. No significant p21 induction occurred in the virus-transformed cell lines. We show that the androgen-independent tumour cell lines are more sensitive to etoposide and vinblastine than the androgen dependent cell line, LNCaP. Except for LNCaP cells, etoposide and vinblastine were found to be three- to ten-fold more effective than estramustine. In the benign hyperplasia cell line, BPH-1, only etoposide is highly effective. Etoposide and vinblastine were found to effectively inactivate the androgen-independent cell lines, in which p53 is dysfunctional.