Jagadananda Ghosh

Inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-Jun N-terminal kinase.

Previously, we reported that inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in both androgen-sensitive (LNCaP) and androgen-refractory (PC3) human prostate cancer cells within hours of treatment [Proc. Natl. Acad. Sci. USA 95 (1998) 13182-13187]. Apoptosis was prevented by exogenous 5(S)-HETE, a product of 5-lipoxygenase, indicating a role of this eicosanoid as an essential survival/anti-apoptotic factor for prostate cancer cells. However, nothing was clearly known about details of the underlying molecular mechanisms or events mediating the induction of fulminating apoptosis in these cells. This report documents the fact that inhibition of arachidonate 5-lipoxygenase induces rapid activation of c-Jun N-terminal kinase (JNK) in human prostate cancer cells which is prevented by the 5-lipoxygenase metabolite, 5(S)-HETE. Activation of JNK is unaffected by the cell-permeable tetra-peptide inhibitors of caspase 8 or caspase 3 (IETD-FMK and DEVD-FMK), though these inhibitors effectively blocked apoptosis triggering, suggesting that activation of JNK is independent or upstream of caspase activation. Both 5-lipoxygenase inhibition-induced activation of JNK and induction of apoptosis are prevented by curcumin, an inhibitor of JNK-signaling pathway. Apoptosis is also blocked by SP600125, a specific inhibitor of JNK activity, indicating that JNK activity is required for the induction of apoptosis in these cells. These findings suggest that the metabolites of arachidonate 5-lipoxygenase promote survival of prostate cancer cells involving down-regulation of stress-activated protein kinase.

Wedelolactone, a medicinal plant-derived coumestan, induces caspase-dependent apoptosis in prostate cancer cells via downregulation of PKCε without inhibiting Akt

Emerging studies indicate that metabolism of arachidonic acid through the 5-lipoxygenase (5-Lox) pathway plays a critical role in the survival of prostate cancer cells raising the possibility that 5-Lox can be targeted for an effective therapy of prostate cancer. Wedelolactone (WDL), a medicinal plant-derived natural compound, is known to inhibit 5-Lox activity in neutrophils. However, its effect on apoptosis in prostate cancer cells has not been addressed. Thus, we tested the effects of WDL on human prostate cancer cells in vitro. We observed that WDL kills both androgen-sensitive as well as androgen-independent prostate cancer cells in a dose-dependent manner by dramatically inducing apoptosis. We also found that WDL-induced apoptosis in prostate cancer cells is dependent on c-Jun N-terminal Kinase (c-JNK) and caspase-3. Interestingly, WDL triggers apoptosis in prostate cancer cells via downregulation of protein kinase Cε (PKCε), but without inhibition of Akt. WDL does not affect the viability of normal prostate epithelial cells (PrEC) at doses that kill prostate cancer cells, and WDL-induced apoptosis is effectively prevented by 5-oxoETE, a metabolite of 5-Lox (but not by 15-oxoETE, a metabolite of 15-Lox), suggesting that the apoptosis-inducing effect of WDL in prostate cancer cells is mediated via inhibition of 5-Lox activity. These findings indicate that WDL selectivity induces caspase-dependent apoptosis in prostate cancer cells via a novel mechanism involving inhibition of PKCε without affecting Akt and suggest that WDL may emerge as a novel therapeutic agent against clinical prostate cancer in human.

Inhibition of 5-lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon

Previous studies have shown that human prostate cancer cells constitutively generate 5-lipoxygenase (5-LOX) metabolites from arachidonic acid, and inhibition of 5-LOX blocks production of 5-LOX metabolites and triggers apoptosis in prostate cancer cells. This apoptosis is prevented by exogenous metabolites of 5-LOX, suggesting an essential role of 5-LOX metabolites in the survival of prostate cancer cells. However, downstream signaling mechanisms which mediate the survival-promoting effects of 5-LOX metabolites in prostate cancer cells are still unknown. Recently, we reported that MK591, a specific inhibitor of 5-LOX activity, induces apoptosis in prostate cancer cells without inhibition of Akt, or ERK, two well-characterized regulators of pro-survival mechanisms, suggesting the existence of an Akt and ERK-independent survival mechanism in prostate cancer cells regulated by 5-LOX. Here, we report that 5-LOX inhibition-induced apoptosis in prostate cancer cells occurs via rapid inactivation of protein kinase C-epsilon (PKCε), and that exogenous 5-LOX metabolites prevent both 5-LOX inhibition-induced down-regulation of PKCε and induction of apoptosis. Interestingly, pre-treatment of prostate cancer cells with diazoxide (a chemical activator of PKCε), or KAE1-1 (a cell-permeable, octa-peptide specific activator of PKCε) prevents 5-LOX inhibition-induced apoptosis, which indicates that inhibition of 5-LOX triggers apoptosis in prostate cancer cells via down-regulation of PKCε. Altogether, these findings suggest that metabolism of arachidonic acid by 5-LOX activity promotes survival of prostate cancer cells via signaling through PKCε, a pro-survival serine/threonine kinase.

Inhibition of 5-lipoxygenase selectively triggers disruption of c-Myc signaling in prostate cancer cells.

Background: Frequent overactivation of c-Myc aggravates a spectrum of cancers. However, cancer-specific inhibition of c-Myc activity poses significant difficulty. Results: Inhibition of 5-lipoxygenase down-regulates c-Myc expression and triggers proteasomal degradation in cancer cells but not in normal fibroblasts. Conclusion: Inhibition of 5-lipoxygenase selectively disrupts c-Myc function in cancer cells. Significance: Inhibition of 5-lipoxygenase may be a novel strategy to control oncogenic c-Myc signaling. Myc is up-regulated in almost all cancer types and is the subject of intense investigation because of its pleiotropic effects controlling a broad spectrum of cell functions. However, despite its recognition as a stand-alone molecular target, development of suitable strategies to block its function is hindered because of its nonenzymatic nature. We reported earlier that arachidonate 5-lipoxygenase (5-Lox) plays an important role in the survival and growth of prostate cancer cells, although details of the underlying mechanisms have yet to be characterized. By whole genome gene expression array, we observed that inhibition of 5-Lox severely down-regulates the expression of c-Myc oncogene in prostate cancer cells. Moreover, inhibition of 5-Lox dramatically decreases the protein level, nuclear accumulation, DNA binding, and transcriptional activities of c-Myc. Both the 5-Lox inhibition-induced down-regulation of c-Myc and induction of apoptosis are mitigated when the cells are treated with 5-oxoeicosatetraenoic acid, a metabolite of 5-Lox, confirming a role of 5-Lox in these processes. c-Myc is a transforming oncogene widely expressed in prostate cancer cells and maintains their transformed phenotype. Interestingly, MK591, a specific 5-Lox inhibitor, strongly affects the viability of Myc-overactivated prostate cancer cells and completely blocks their invasive and soft agar colony-forming abilities, but it spares nontransformed cells where expression of 5-Lox is undetectable. These findings indicate that the oncogenic function of c-Myc in prostate cancer cells is regulated by 5-Lox activity, revealing a novel mechanism of 5-Lox action and suggesting that the oncogenic function of c-Myc can be suppressed by suitable inhibitors of 5-Lox.

OXER1, a G protein-coupled oxoeicosatetraenoid receptor, mediates the survival-promoting effects of arachidonate 5-lipoxygenase in prostate cancer cells

Inhibition of 5-Lox induces apoptosis in prostate cancer cells by inactivating PKCε which is prevented by 5-oxoETE, and activators of PKCε prevent 5-Lox inhibition-induced apoptosis, suggesting that 5-Lox metabolites exert survival signaling via PKCε. However, mechanisms by which 5-Lox metabolites activate PKCε are not understood yet. We found that prostate cancer cells express high levels of OXER1, a G protein-coupled 5-oxoETE receptor, which delivers signal by generating diacyl-glycerol through phospholipase C-beta. Interestingly, we found that U73122, an inhibitor of PLC-beta, interrupts the apoptosis-preventing effect of 5-oxoETE, and exogenous diacyl-glycerol effectively prevents 5-Lox inhibition-induced apoptosis, suggesting that 5-oxoETE signals via OXER1 to promote prostate cancer cell survival.

Wedelolactone, an anti-inflammatory botanical, interrupts c-Myc oncogenic signaling and synergizes with enzalutamide to induce apoptosis in prostate cancer cells

The c-Myc gene encodes an oncoprotein transcription factor that is frequently upregulated in almost all cancer types and is the subject of intense investigation for management of cancer because of its pleiotropic effects controlling a spectrum of cellular functions. However, due of its nonenzymatic nature, development of suitable strategies to block its protein–protein or protein–DNA interaction is challenging. Thus, c-Myc has been recognized as an elusive molecular target for cancer control, and various approaches are in development to inhibit c-Myc transcriptional activity. We observed that wedelolactone (WDL), an anti-inflammatory botanical compound, severely downregulates the expression of c-Myc mRNA in prostate cancer cells. Moreover, WDL dramatically decreases the protein level, nuclear accumulation, DNA-binding, and transcriptional activities of c-Myc. c-Myc is a transforming oncogene widely expressed in prostate cancer cells and is critical for maintaining their transformed phenotype. Interestingly, WDL was found to strongly affect the viability of Myc-activated prostate cancer cells and completely block their invasion as well as soft agar colony formation in vitro. WDL was also found to downregulate c-Myc in vivo in nude mice xenografts. Moreover, WDL synergizes with enzalutamide to decrease the viability of androgen-sensitive prostate cancer cells via induction of apoptosis. These findings reveal a novel anticancer mechanism of the natural compound WDL, and suggest that the oncogenic function of c-Myc in prostate cancer cells can be effectively downregulated by WDL for the development of a new therapeutic strategy against Myc-driven prostate cancer. Mol Cancer Ther; 15(11); 2791–801. ©2016 AACR.

MK591, a second generation leukotriene biosynthesis inhibitor, prevents invasion and induces apoptosis in the bone-invading C4-2B human prostate cancer cells: Implications for the treatment of castration-resistant, bone-metastatic prostate cancer

Castration-resistant prostate cancer (CRPC) is a major clinical challenge for which no cure is currently available primarily because of the lack of proper understanding about appropriate molecular target(s). Previously we observed that inhibition of 5-lipoxygenase (5-Lox) activity induces apoptosis in some types of prostate cancer cells, suggesting an important role of 5-Lox in the viability of prostate cancer cells. However, nothing is known about the role of 5-Lox in the survival of castration-resistant, metastatic prostate cancer cells. Thus, we tested the effects of MK591, a second-generation, specific inhibitor of 5-Lox activity, on the viability and metastatic characteristics of CRPC cells. We observed that MK591 effectively kills the bone-invading C4-2B human prostate cancer cells (which bear characteristics of CRPC), but does not affect normal, non-cancer fibroblasts (which do not express 5-Lox) in the same experimental conditions. We also observed that MK591 dramatically inhibits the in vitro invasion and soft-agar colony formation of C4-2B cells. Interestingly, we found that treatment with MK591 dramatically down-regulates the expression of c-Myc and its targets at sub-lethal doses. In light of frequent over-activation of c-Myc in a spectrum of aggressive cancers (including CRPC), and the challenges associated with inhibition of c-Myc (because of its non-enzymatic nature), our novel findings of selective killing, and blockade of invasive and soft-agar colony-forming abilities of the castration-resistant, bone-metastatic C4-2B prostate cancer cells by MK591, open up a new avenue to attack CRPC cells for better management of advanced prostate cancer while sparing normal, non-cancer body cells.

Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase

The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44 (+), CD133 (+) cell subpopulations were isolated from human prostate tumors which exhibit stem-like properties showing therapeutic-resistance, capacity of self-renewal, and exact recapitulation of the original tumor in vivo. Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44 (+), CD133 (+) cell subpopulations were isolated from human prostate tumors which exhibit stem-like properties showing therapeutic-resistance, capacity of self-renewal, and exact recapitulation of the original tumor in vivo. Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.

Abstract 2253: Wedelolactone, a medicinal plant-derived coumestan, induces caspase-dependent apoptosis in prostate cancer cells via down-regulation of PKC-epsilon without inhibiting Akt.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Prostate cancer cells continuously generate 5(S)-HETE series of metabolites via 5-lipoxygenase (5-Lox) activity, and inhibition of 5-Lox blocks production of metabolites and triggers apoptosis both in androgen-sensitive and androgen-independent prostate cancer cells. Apoptosis is prevented by 5(S)-HETE and 5-oxoETE, but not by leukotrienes or metabolites of 12-Lox or 15-Lox, which is consistent with the idea that the 5-HETE series of metabolites are essential survival factors for prostate cancer cells. Recently, we found that 5-Lox-regulated survival-signaling is mediated via protein kinase C-epsilon (PKCe), but not Akt, or ERK. Prostate tumor tissues express high levels of 5-Lox, but the expression of 5-Lox in normal prostate glands is undetectable. Thus, 5-Lox emerges as a novel molecular target for prostate cancer therapy. However, lack of suitable 5-Lox inhibitors for in vivo use is hampering progress towards clinical development. Several natural compounds possess 5-Lox inhibitory effects, raising the possibility that effective 5-Lox inhibitors may be found in natural products for use against prostate cancer. Wedelolactone (WDL), a medicinal plant-derived coumestan, is known to inhibit 5-Lox activity in neutrophils. However, its potential on apoptosis induction in prostate cancer cells is not known. Thus, we tested the effects of WDL on a range of human prostate cancer cells in vitro. Methods: Prostate cancer cells were treated with doses of WDL for varying periods of time. Apoptosis was measured by annexin-V binding, PARP-cleavage, and DNA-degradation. Role of caspase in apoptosis was tested by enzymatic assays and chemical inhibitors. Effects of WDL on PKC-epsilon and Akt were analyzed by Western blot and enzymatic assays. Results: We observed that WDL kills both androgen-sensitive as well as androgen-independent prostate cancer cells in a dose-dependent manner by dramatically inducing apoptosis. This apoptosis is dependent on c-Jun N-terminal Kinase (c-JNK) and caspase-3. Interestingly, WDL was observed to trigger apoptosis in prostate cancer cells via down-regulation of protein kinase C-epsilon (PKCe), but without any inhibition of Akt. WDL does not affect the viability of normal prostate epithelial cells (PrEC) at doses that kill prostate cancer cells. Moreover, it was found that WDL-induced apoptosis is effectively prevented by 5-oxoETE, a metabolite of 5-Lox, confirming that WDL induces apoptosis in prostate cancer cells via inhibition of 5-Lox activity. Conclusion: Our findings indicate that WDL selectivity induces caspase-dependent apoptosis in prostate cancer cells via a novel mechanism involving inhibition of the oncogenic serine/threonine kinase PKCe without affecting Akt, and suggest that WDL may emerge as a novel therapeutic agent against clinical prostate cancer in human. Citation Format: Sivalokanathan Sarveswaran, Subhash Gautam, Jagadananda Ghosh. Wedelolactone, a medicinal plant-derived coumestan, induces caspase-dependent apoptosis in prostate cancer cells via down-regulation of PKC-epsilon without inhibiting Akt. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2253. doi:10.1158/1538-7445.AM2013-2253

Abstract 2702: Suppression of c-Myc oncogenic signaling by MK591, an inhibitor of arachidonate 5-lipoxygenase

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Prostate cancer cells continuously generate 5(S)-HETE series of metabolites from arachidonic acid via 5-lipoxygenase (5-Lox) activity, and inhibition of 5-Lox blocks production of metabolites and triggers apoptosis both in androgen-sensitive as well as androgen-independent prostate cancer cells. This apoptosis is prevented by 5(S)-HETE and 5-oxoETE, but not by leukotrienes or metabolites of 12-Lox or 15-Lox, which is consistent with the idea that the 5-HETE series of metabolites are essential survival factors for prostate cancer cells. Thus, 5-Lox emerges as a novel molecular target for prostate cancer therapy. However, details of the downstream signaling mechanisms regulated by 5-Lox metabolites in prostate cancer cells are yet to be fully characterized, and the lack of suitable compounds with in vivo efficacy is hampering proper translational development to target 5-Lox. We addressed this problem by treating LNCaP human prostate cancer cells with MK591, a specific, third-generation inhibitor of 5-Lox activity, and analyzing the effects by gene-expression array, RT-PCR, and Western blot. Methods: Cells were treated with MK591 for eight hours and gene expression was analyzed by Illumina HT-12v4 whole genome gene-expression array. Expression of c-Myc was confirmed by RT-PCR and Western blot. Apoptosis was measured by annexin-V binding, PARP-cleavage, and detecting degradation of chromatin-DNA to nucleosomes. Transcriptional activation of c-Myc was analyzed by nuclear localization, DNA-binding, luciferase-reporter assays and expression of c-Myc-target genes. Results: We observed that MK591 strongly inhibits the expression of c-Myc oncogene in prostate cancer cells, as revealed by the gene expression array. Down-regulation of c-Myc by MK591 was confirmed by RT-PCR and Western blot as well as by the reduction in nuclear-localization and DNA-binding activities of c-Myc proteins. Treatment with MK591 dramatically decreased the Myc-driven E-box-luciferase reporter activity in prostate cancer cells which is effectively prevented by 5-oxoETE, a metabolic product of 5-Lox. Moreover, MK591 substantially reduced the expression of c-Myc target genes (e.g., cyclin D1, CDK4, survivin, Aurora kinase, Gemin4 and MCM10), and blocked the matrigel-invasion and soft-agar colony-formation by prostate cancer cells. Interestingly, while MK591 dramatically affected the Myc function in prostate cancer cells (where Myc is over-activated), it did not inhibit the basal c-Myc functions in non-cancer cells, such as human foreskin fibroblasts (HFF) which do not expresses 5-Lox. Conclusion: Our findings indicate that expression and oncogenic-function of c-Myc in prostate cancer cells are severely down-regulated by the 5-Lox inhibitor, MK591, and suggest that MK591 may be useful to treat advanced, aggressive prostate cancers which are frequently associated with over-activation of the c-Myc oncogene. Citation Format: Sivalokanathan Sarveswaran, Debrup Chakraborty, Jagadananda Ghosh. Suppression of c-Myc oncogenic signaling by MK591, an inhibitor of arachidonate 5-lipoxygenase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2702. doi:10.1158/1538-7445.AM2014-2702