James Kumi-Diaka

The mediating role of caspase‐3 protease in the intracellular mechanism of genistein‐induced apoptosis in human prostatic carcinoma cell lines, DU145 and LNCaP

Summry— A series of in vitro studies were carried out to investigate genistein‐induced cell death, and the nature of cell death, in two human prostate cancer cell lines (LNCaP and Du145), and the possible involvement of caspase‐3 protease in genistein‐induced apoptosis in the target cells. The major findings of these studies are: i) genistein inhibits growth and proliferation of both LNCaP and DU145 cells via apoptosis mainly, and necrosis at higher concentrations; ii) genistein induces activation and expression of caspase‐3 (CPP32) in both target cells; iii) genistein‐induced apoptosis and CPP32 activation could be significantly inhibited by the caspase‐3 inhibitor, z‐VAD‐fmk (N—benzyloxycarbonyl‐Val‐Asp‐fluoromethyl—ketone), thus confirming a mediator role of CPP32 in the genistein‐induced apoptotic pathway in the target cells. The potency of most known chemopreventive drugs for cancer is due to induction of apoptosis in solid tumors (Thompson, Science 267 (1995) 1456; Gurney et al., Science 288 (2000) 283). Inevitably, agents that increase transcription of caspase‐3 protease could reinforce cell death via CPP32‐mediated apoptosis. In this regard, genistein may find an application in the treatment of human prostate carcinoma, independently of hormone sensitivity.

Cytotoxic potential of the phytochemical genistein isoflavone (4',5',7-trihydroxyisoflavone) and certain environmental chemical compounds on testicular cells

Summry— The effects of genistein (Gn), sodium azide (naz), and dexamethasone (dxm) on testicular cells TM3, TM4 and GC‐1 spg were studied in vitro. First, a series of experiments were performed to assess the response of the cells to the exposure of Gn, naz, dxm, a combination of Gn with naz and Gn with dxm. Trypan blue exclusion assay was used to determine the percentage of viability, and LDH‐cytotoxicity test was used to assess the degree of treatment‐induced cytotoxicity on each cell type. A second series of experiments were performed to study cytomorphology and determine the type and percentage of treatment‐induced cell death (apoptosis and necrosis) on each cell line, using fluorescent dye technique to detect apoptotic and necrotic cells, and tunnel assay to confirm apoptosis. The results from the data obtained demonstrated: i) that incubation of testis cells with each of the agents (Gn, dxm, naz) alone and in two combinations (Gn‐dxm, and Gn‐naz) induced significant testicular cell death; ii) that both genistein and dexamethasone mostly and significantly induced apoptotic cell death while sodium azide induced necrotic cell death; iii) that addition of dexamethasone to genistein demonstrated synergism in apoptosis on testis cells; and iv) that combination of naz with Gn demonstrated synergism in necrosis on testis cells even though Gn alone did not induce significant necrosis. It is concluded that the synergistic actions of genistein and dxm, and of genistein + sodium azide in induction of apoptosis and/or necrosis may be of clinical and pathophysiological research interest considering the chemopreventive and chemotherapeutic potential of genistein; and the clinico‐pharmacological application of dexamethasone and sodium azide.

Caspase-3 protease activation during the process of genistein-induced apoptosis in TM4 testicular cells

Summary— The role of caspase‐3 (CPP32) protease in the molecular pathways of genistein‐induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst‐33258‐PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3‐[4,5‐dimethyl‐thiazol‐2‐yl]‐2,5‐diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase‐3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose‐ and time‐dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase‐3 enzymatic activity; iv) genistein‐induction of apoptosis and necrosis was significantly inhibited by the caspase‐3 inhibitor, z‐DEV‐FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein‐induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase‐3 (CPP332) mediation in the molecular pathways of genistein‐induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.

Influence of genistein (4',5,7-trihydroxyisoflavone) on the growth and proliferation of testicular cell lines.

The effects of genistein on testicular cells, TM3, TM4, and GC-1 spg, were studied in vitro. First, each cell line was cultured with pre-determined concentrations of genistein for a maximum of 72 h to assess the effects of genistein on in vitro growth of the test cells. A second series of experiments were performed to determine the degree of genistein-induced apoptosis in these cells, using Apop-Tag kit reagents, to detect apoptotic cells in situ by specific end labeling, and detection of DNA fragments produced by the apoptotic process. The results obtained indicate that: i) genistein inhibits the growth and proliferation of testicular cells; ii) growth inhibition and proliferation is dose- and exposure-time dependent; iii) there is significant difference in sensitivity of the different testicular cells to genistein; iv) genistein induces apoptosis in testicular cells in a concentration-dependent manner. Genistein-induced apoptosis identifies genistein as a potential diagnostic and therapeutic tool in testicular pathophysiological research.

Potential mechanism of phytochemical-induced apoptosis in human prostate adenocarcinoma cells: Therapeutic synergy in genistein and β-lapachone combination treatment

BackgroundProstate cancer is the second leading cause of male death in the United States. The incidence increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of the cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited; and hence treatment has only been partially successful. The objective of the current studies was to examine the role of caspase 3 (CPP32) and NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of genistein-and β-lapachone (bLap)-induced apoptosis in human prostate carcinoma cells PC3.ResultsBoth genistein and bLap produced dose-dependent growth inhibition and treatment-induced apoptosis in PC3. Treatment with caspase 3 inhibitor, DEVD-fmk before exposure to genistein, significantly inhibited caspase 3 expression and treatment-induced apoptosis; implicating CPP32 as the main target in genistein-induced apoptosis in PC3. Contrary to this observation, inhibition of CPP32 did not significantly influence bLap-induced apoptosis; implying that the major target of bLap-induced apoptosis may not be the caspase. Treatment with NQO1 inhibitor, dicoumarol (50 μM), prior to exposure of PC3 to bLap led to significant decrease in bLap toxicity concurrent with significant decrease in treatment-induced apoptosis; thus implicating NQO1 as the major target in β-lapachone-induced apoptosis in PC3. In addition, the data demonstrated that NQO1 is the major target in bLap-genistein (combination)-induced apoptosis. On the contrary, blocking NQO1 activity did not significantly affect genistein-induced apoptosis; implying that NQO1 pathway may not be the main target for genistein-induced apoptosis in PC3 cells. Furthermore, blocking NQO1 and CPP32 did not confer 100% protection against genistein-induced or bLap-induced apoptosis.ConclusionThe data thus demonstrate that both genistein-and bLap-induced apoptosis are mostly but not completely dependent on CPP32 and NQO1 respectively. Other minor alternate death pathways may be involved. This suggests that some death receptor signals do not utilize the caspase CPP32 and/or the NQO1 death pathways in PC3. The demonstrated synergism between genistein and bLap justifies consideration of these phytochemicals in chemotherapeutic strategic planning.

Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and β-lapachone

A wide spectrum of anti‐cancer activity of genistein and β‐lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and β‐lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3‐〚4,5‐dimethylthiazol‐2‐yl〛‐2‐,5‐diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment‐induced growth inhibition and cytotoxicity and, annexin V‐fluoresceine (FI) and terminal deoxyribonucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL)–propidium iodide (PI) assays to determine potential treatment‐induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and β‐lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment‐induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with β‐lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or β‐lapachone alone. β‐lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2–M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether β‐lapachone—genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.

ANTICANCER ACTIVITIES OF GENISTEIN-TOPOTECAN COMBINATION IN PROSTATE CANCER CELLS

Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7‐trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti‐tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA‐approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein‐topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO2 for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein‐topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post‐treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment‐induced apoptosis using caspase‐3 and ‐9 binding assays and (iii) treatment‐induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein‐topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase‐3 and ‐9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein‐topotecan combination treatment. Treatments involving genistein‐topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.

Genistein-selenium combination induces growth arrest in prostate cancer cells.

The prognosis for patients with metastasized prostate cancer is still poor, despite conventional aggressive therapeutic modalities. Several in vitro studies together with animal models and epidemiological studies have indicated that phytochemicals can be antitumorigenic and may be protective against human cancers. However, the potential antitumor effects of genistein isoflavone, a widely studied nutrient phytochemical, have been equivocal. In this study, we investigated the effects of genistein-selenium (Gn-Se) combination on chemosensitivity and matrix metalloproteinase-2 (MMP-2) expression levels in PC3 (hormone-independent) and LNCaP (hormone-dependent) prostate cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium staining and ATP bioassay showed that genistein, selenium, and Gn-Se combination significantly inhibited growth of LNCaP and PC3 cells in a dose- and time-dependent manner, independent of hormonal status, and with no significant differences in chemosensitivity between LNCaP and PC3. Gn-Se combination induced significantly the greatest growth inhibition in both cell lines. Growth inhibition was through apoptosis induction. The treatment-induced apoptotic cascades are caspase-dependent, with evidence of an alternative non-caspase pathway(s). Treatment also induced a dose- and time-dependent decrease in MMP-2 expression levels in PC3 and LNCaP with no significant differences between the two cells. Gn-Se combination induced the greatest depression in MMP-2. Overall, none of the treatment modalities had any significant inhibitory effect in normal prostate epithelial cells. The data obtained from the present study indicate that Gn-Se combination may have chemopreventive value and/or may be adjuvant to standard therapy for prostate tumors independent of hormonal status. MMP-2 expression in cancer cells has been associated with active invasion and metastasis.

Abstract 5422: Synergistic effects of metabolic inhibitors on radiochemosensitized spheroid prostate cancer cells

One of the ways by which prostate cancer cells survive or become resistant to conventional therapies is through the ability to reprogram their metabolic pathways. Thus, justifying the need for studies to elucidate the efficacy of metabolic inhibitors in prostate cancer intervention. The purpose of this study was to investigate the synergistic effects of two metabolic pathway inhibitors - 3-bromopyruvate (3-BrPA) and genistein - on androgen sensitive (LNCaP) and androgen insensitive (DU145) spheroid prostate cancer cells separately primed or irradiated with very low doses of x-ray radiation (VLDR) and low frequency weak magnetic field radiation (MFR). Methods: Both PCa cells were cultured until they form spheroids and then irradiated with either VLDR (20mGy/hr) or MFR (10mT; 50Hz/hr) in separate groups. Cells were seeded and treated with varying doses of 3-BrPA and genistein in single or combination regimen. Cell viability, metabolic activity and cytotoxicity or LDH release levels were measured using MTT, Alamar blue and LDH assays respectively. NBT assay was used to determine treatment-induced ROS levels. mitochondrial membrane potential was determined by Rh123 and ethidium bromide fluorescent staining. Modes of Cell death were determined by fluorescent staining with acridine orange and ethidium bromide dyes, and by morphological identification of apoptotic and autophagic PCa cells. CompuSyn program was used to determine the time- and dose-dependent synergism between combination regimens. Results: Our results show that VLDR and weak MFR moderately inhibit cell proliferation in both PCa cell lines. However, LNCaP PCa cells were found to be more responsive to both VLDR or weak MFR compared to DU-145 PCa cells. Both VLDR and MFR suppresses ATP generation and increases ROS generation time-dependently in both cell lines. The most common cell death event found in MFR-exposed cells was a mixture of mitotic catastrophe and autophagy, while autophagic and slight apoptotic responses were observed in VLDR-exposed cells. 3-BrPA triggers more autophagic response in irradiated DU145 cells compared more apoptotic response in LNCaP cells. In contrast, genistein induced cell growth inhibition in both cell lines, but more effect was observed in LNCaP cells with dose-dependent apoptotic response. Combination of 3-BrPA and genistein forced PCa cells to undergo reprogrammed cell death, decreased the mitochondrial membrane potential and LDH levels significantly in a dose and time-dependently. Conclusion: The combination treatments show synergistic and significant inhibition of the glycolytic and mitochondrial metabolic pathways resulting in induction of apoptosis in radiochemosensitized PCa cells. Priming of cells with VLDR or MFR also enhanced the therapeutic effects of both metabolic inhibitors. Hence, reinforcing the potential benefit of metabolic inhibitors in prostate cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Saheed O. Oseni, Rolando Branly, Mirjana Pavlovic, James Kumi-Diaka. Synergistic effects of metabolic inhibitors on radiochemosensitized spheroid prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2017-5422

Abstract C85: Novel molecular targets for genistein in prostate cancer cells

Background: Prostate cancer is the most common form of non‐skin cancer and the second leading cause of cancer deaths within the United States. The five year survival rate has increased from 69% to 99% over the last 25 years for the local and regional disease, but has remained fairly low (approximately 34%) for the advanced disease. Current research is aimed at finding complementary or alternative treatments that will specifically target components of signal transduction, cell‐cycle and apoptosis pathways to induce cell death, with little or no toxic‐side effects to the patient. In this study we investigated the effect of genistein on expression levels of genes involved in these pathways. Genistein (4, 5, 7‐trihydroxy‐isoflavone) is a major isoflavone micronutrient constituent of soy that has been shown to inhibit growth proliferation and induce apoptosis in cancer cells. Methods: The mechanism of genistein‐induced cell death and potential molecular targets for genistein in LNCaP prostate cancer cells were investigated using several techniques. The chemosensitivity of genistein towards prostate cancer cells was investigated using the ATP and MTS assays and apoptosis induction was determined using apoptosis indicator assays and caspase assays. Expression levels of several molecular targets were also determined using cDNA microarray and RT‐PCR analysis. Results: Our results revealed that genistein induces cell death in a time and dose‐dependent manner and regulates expression levels of several genes involved in carcinogenesis and immunogenicity. Several cell cycle genes were down‐regulated including the mitotic kinesins, cyclins and cyclin dependent kinases, indicating that genistein is able to halt cell cycle progression through the regulation of genes involved in this process. Several members of the Bcl‐2 family which are involved in apoptosis were also affected and a number of genes involved in immunogenicity were upregulated including DefB1 and HLA membrane receptors. Conclusion: The results of this study provide evidence of genistein9s ability to inhibit cancer cell proliferation and induce apoptosis which indicates its potential as an adjuvant in chemotherapy and immunotherapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C85.