Anti-cancer activity of withaferin A in B-cell lymphoma.

Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90.

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells.

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.

Cancer-selective apoptotic effects of extracellular and intracellular Par-4.

Selectivity toward cancer cells is the most desirable element in cancer therapeutics. Par-4 is a cancer cell-selective proapoptotic protein that functions intracellularly in the cytoplasmic and nuclear compartments as a tumor suppressor. Moreover, recent findings indicate that the Par-4 protein is secreted by cells, and extracellular Par-4 induces cancer cell-specific apoptosis by interaction with the cell-surface receptor GRP78. This review describes the mechanisms underlying the apoptotic effects of both extracellular and intracellular Par-4 acting through its effector domain SAC.

Par-4 drives trafficking and activation of Fas and Fasl to induce prostate cancer cell apoptosis and tumor regression.

Prostate cancer cells are generally resistant to apoptosis by conventional therapy. During a search for molecules that may overcome prostate cancer cell survival mechanisms, we identified the prostate apoptosis response-4 (Par-4) gene. Par-4 induced apoptosis of selective prostate cancer cells PC-3, DU-145, and TSU-Pr and caused tumor regression by inhibition of NF-kappaB activity and cell membrane trafficking of Fas and FasL that leads to the activation of the Fas-Fas-associated death domain-caspase-8 pro-death pathway. Neither Fas pathway activation alone nor inhibition of NF-kappaB activity with IkappaB-super repressor was sufficient to induce apoptosis of prostate cancer cells. Coregulation of these two pathways was essential and sufficient for Par-4 to induce apoptosis. On the other hand, prostate cancer cells LNCaP or normal prostatic epithelial cells that were resistant to apoptosis by Par-4 did not show Fas or FasL trafficking. These findings identify a mechanism of apoptosis by Par-4 and suggest that Par-4 may have therapeutic potential.

TPA-activated transcription of the human MnSOD gene: role of transcription factors Sp-1 and Egr-1.

Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription.

Differential expression levels of Par-4 in melanoma.

The pro-apoptotic prostate apoptosis response-4 gene product Par-4 sensitizes prostate cells to the induction of programmed cell death. In this study we examined Par-4 expression in human melanoma cell lines and melanoma metastases. The heterogeneous expression detected prompted us to investigate the biological relevance of Par-4 in a human melanoma xenotransplantation model. Overexpression of Par-4 by transfection decreased tumour development in xenotransplanted A375-C6 melanoma cells in SCID mice and correlated to an increase in tumour cell apoptosis. These data suggest that high expression of the pro-apoptotic protein Par-4 could qualify as a prognostic marker in human melanoma.

Regional expression of Par-4 mRNA and protein after fluid percussion brain injury in the rat.

Regional levels of prostate apoptosis response-4 (Par-4) protein and mRNA were measured after lateral fluid percussion (FP) brain injury in rats. Immunochemical studies indicated that Par-4 immunoreactivity (ir) is present in cortical neurons and hippocampal CA1-CA3 pyramidal neurons in uninjured rats. Increases of Par-4-ir were observed in the CA3 neurons of the ipsilateral hippocampus (IH), but not in injured left cortex (IC) at 48 h after FP brain injury. Levels of the Par-4 mRNA measured by RT-PCR assay and protein measured by Western blot procedure were significantly increased in the injured IC and IH, but not in the contralateral right cortex and hippocampus after brain injury. Levels of both Par-4 protein and mRNA were significantly increased in the IC and IH as early as 2 h and stayed elevated at 24 and 48 h after injury. These data show that the induction of proapoptotic Par-4 mRNA and protein occurs only in the IC and IH that have been observed to undergo apoptosis and neuronal cell loss after lateral FP brain injury. Because increased expression of Par-4 has been observed to contribute to apoptosis and cell death in cultured neurons, the present temporal pattern of Par-4 expression is consistent with a role for Par-4 in apoptosis and neuronal cell death after traumatic brain injury.

Deregulated expression of prostate apoptosis response gene-4 in less differentiated lymphocytes and inverse expressional patterns of par-4 and bcl-2 in acute lymphocytic leukemia.

INTRODUCTION: Prostate apoptosis response gene-4, known as par-4, is a new proapoptotic factor functionally required but not sufficient for apoptosis. Since there is evidence from prostate cancer cells that par-4 is involved in regulation of bcl-2 we assessed expression of par-4 and bcl-2 in different populations of normal and neoplastic lymphocytes. MATERIALS AND METHODS: Expression of par-4 mRNA and protein in different subpopulations of normal and neoplastic lymphocytes was assessed by reverse transcription polymerase chain reaction and Western blot. RESULTS: Par-4 mRNA was not detectable in lymphocytes of healthy volunteers (n = 10), but was present in the majority of samples of chronic lymphocytic leukemia (n = 30), chronic lymphocytic leukemia/prolymphocytic leukemia (n = 6) and acute lymphocytic leukemia (n = 10). Par-4 protein was expressed unanimously in samples of mononuclear cells from healthy volunteers and patients with CLL, but less frequently in immature lymphocytes, including neoplastic cells of CLL/PLL and ALL. The decreased frequency of par-4 expression in immature subpopulations was confirmed by results on lymphocytic cell lines at various stages of maturation. Comparing the expressional patterns of par-4 and bcl-2 there was an inverse relationship of both proteins in ALL and different lymphocytic cell lines, indicating a functional relationship of par-4 and bcl-2. CONCLUSIONS: This study establishes par-4 as a factor expressed in the majority of normal and neoplastic lymphocytic cells, demonstrating a decreased frequency of protein expression in less differentiated lymphocytes and an inverse expressional pattern of par-4 and bcl-2 in lymphocytic cell lines and ALL.

Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis.

In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1(-/-)) and heterozygous (Egr-1(+/-)) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1(+/-) cells (22.8%; p < 0.0001) when compared with Egr-1(-/-) cells (3.5%). Radiation elevated p53 protein in Egr-1(+/-) cells in 3-6 h. However, in Egr-1(-/-) cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1(+/-) cells but not in Egr-1(-/-) cells. Interestingly, transient overexpression of EGR-1 in p53(-/-) MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53(+/+) MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed in Egr-1(-/-) MEF cells before and after radiation when compared with the levels observed in Egr-1(+/-) cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1(-/-) cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1(-/-) MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53.

Regulation of apoptosis in prostate cancer.

Transformation and malignant progression of prostate cancer is regulated by the inability of prostatic epithelial cells to undergo apoptosis rather than by increased cell proliferation. The basic apoptotic machinery of most prostate cancer cells is intact and the inability to undergo apoptosis is due to molecular alterations that result in failure to initiate or execute apoptotic pathways. This review discusses the role of anti-apoptotic proteins such as Bcl-2/BclXL, NF-kappaB, IGF, caveolin, and Akt, and pro-apoptotic molecules such as PTEN, p53, Bin1, TGF-beta, and Par-4 that can regulate progression of prostate cancer. In addition to highlighting the salient features of these molecules and their relevance in apoptosis, this review provides an appraisal of their therapeutic potential in prostate cancer. Molecular targeting of these proteins and/or their innate pro- or anti-apoptotic pathways, either singly or in combination, may be explored in conjunction with conventional and currently available experimental strategies for the treatment of both hormone-sensitive and hormone-resistant prostate cancer.

The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2.

This study investigates the apoptotic activity of the cyclooxygenase-2 (COX-2) inhibitor celecoxib in prostate carcinoma cells. COX-2 is constitutively expressed in androgen-responsive LNCaP and androgen-nonresponsive PC-3 cells. Exposure of these cells to celecoxib induces characteristic features of apoptosis, including morphological changes, DNA laddering, and caspase-3 activation, whereas piroxicam, a COX-1-specific inhibitor, displays no appreciable effect on either cancer cell line even after prolonged exposure. Moreover, the potency of celecoxib in apoptosis induction is significantly higher than that of other COX-2 inhibitors examined despite the observation that these inhibitors exhibit similar IC(50) in COX-2 inhibition. It is noteworthy that normal human prostate epithelial cells, expressing a marginally detectable level of COX-2, are insensitive to the induction of apoptosis by celecoxib. These data suggest a correlation between COX-2 expression and sensitivity to the apoptotic effect of the COX-2 inhibitor. In an effort to delineate the underlying mechanism, we examined the effect of celecoxib on the expression of Bcl-2 as well as the activation of the key anti-apoptotic kinase Akt. In contrast to an earlier report that attributed the apoptotic activity of NS398 in LNCaP cells to Bcl-2 down-regulation, we provide evidence that the induction of apoptosis by celecoxib in LNCaP and PC-3 cells is independent of Bcl-2. First, treatment with celecoxib does not alter the cellular Bcl-2 level in both cell lines. Second, enforced Bcl-2 expression in PC-3 cells does not confer protection against the induction of apoptosis by celecoxib. Our data show that celecoxib treatment blocks the phosphorylation of Akt. This correlation is supported by studies showing that overexpression of constitutively active Akt protects PC-3 cells from celecoxib-induced apoptosis. Nevertheless, how celecoxib down-regulates Akt is not clear because the drug does not adversely affect phosphoinositide 3-kinase activity in vivo and okadaic acid, a protein phosphatase 2A inhibitor, cannot rescue the inhibition. In summary, our data demonstrate that inhibition of Akt activation may play a crucial role in the induction of apoptosis by celecoxib.

Negative regulation of Par-4 by oncogenic Ras is essential for cellular transformation.

Oncogenic variants of the cellular Ras protein are often associated with different types of human cancers. However, the mechanisms by which oncogenic Ras induces transformation are not fully established. Expression of the transcriptional repressor Par-4 was down-regulated by oncogenic Ras via the Raf-MEK-ERK pathway. Restoration of Par-4 levels by abrogation of the Raf-MEK-ERK pathway with the MEK-inhibitor PD98059 or by ectopic Par-4, that acted to inhibit ERK expression and activation, was sufficient to suppress oncogenic Ras-induced transformation. These findings identify Par-4 as a novel target that has to be down-modulated by oncogenic Ras for successful transformation.

Oncogenic Ras sensitizes cells to apoptosis by Par-4.

Certain mutations in the mammalian ras gene are oncogenic and are often detected in human cancers. Oncogenic Ras induces the transcription activity of NF-kappaB that confers cell survival. Oncogenic Ras also down-modulates the expression of Par-4, a transcriptional repressor protein, that is essential but not sufficient on its own to induce apoptosis. Here we show that reintroduction of Par-4 by transient transfection leads to apoptosis in cells expressing oncogenic Ras but not in those that lack oncogenic Ras expression. Par-4 abrogates oncogenic Ras-inducible NF-kappaB transcription activity but does not interfere with cytoplasmic activation, or the DNA binding activity, of NF-kappaB. Because abrogation of NF-kappaB transcription activity is sufficient to cause apoptosis in cells expressing oncogenic Ras, our findings identify Par-4 as a novel example of a pro-apoptotic protein that selectively inhibits oncogenic Ras-dependent NF-kappaB function at the transcription level and suggest a mechanism by which Par-4 expression may selectively induce apoptosis in oncogenic Ras-expressing cells.

Evidence that Par-4 participates in the pathogenesis of HIV encephalitis.

Progressive neuronal degeneration in brain regions involved in learning and memory processes is a common occurrence in patients infected with human immunodeficiency virus type 1 (HIV-1). We now report that levels of Par-4, a protein recently linked to neuronal apoptosis in Alzheimer's disease, are increased in neurons in hippocampus of human patients with HIV encephalitis and in monkeys infected with a chimeric strain of HIV-1 and simian immunodeficiency virus. Par-4 levels increased rapidly in cultured hippocampal neurons following exposure to the neurotoxic HIV-1 protein Tat, and treatment of the cultures with a Par-4 antisense oligonucleotide protected the neurons against Tat-induced apoptosis. Additional findings show that Par-4 participates at an early stage of Tat-induced neuronal apoptosis before caspase activation, oxidative stress, and mitochondrial dysfunction. Our data suggest that Par-4 may be a mediator of neuronal apoptosis in HIV encephalitis and that therapeutic approaches targeting the Par-4 apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in patients infected with HIV-1.

Prostate apoptosis response-4 production in synaptic compartments following apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration.

Synapses are often located at great distances from the cell body and so must be capable of transducing signals into both local and distant responses. Although progress has been made in understanding biochemical cascades involved in neuronal death during development of the nervous system and in various neurodegenerative disorders, it is not known whether such cascades function locally in synaptic compartments. Prostate apoptosis response-4 (Par-4) is a leucine zipper and death domain-containing protein that plays a role in neuronal apoptosis. We now report that Par-4 levels are rapidly increased in cortical synaptosomes and in dendrites of hippocampal neurons in culture and in vivo, following exposure to apoptotic or excitotoxic insults. Par-4 expression is regulated at the translational level within synaptic compartments. Par-4 antisense treatment suppressed mitochondrial dysfunction and caspase activation in synaptosomes and prevented death of cultured hippocampal neurons following exposure to excitotoxic and apoptotic insults. Local translational regulation of death-related proteins in synaptic compartments may play a role in programmed cell death, adaptive remodeling of synapses, and neurodegenerative disorders.

Mutually exclusive expression patterns of Bcl-2 and Par-4 in human prostate tumors consistent with down-regulation of Bcl-2 by Par-4.

Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis. Constitutive- or regulated- overexpression of Par-4 caused a reduction in the levels of the anti-apoptotic protein Bcl-2. Replenishment of Bcl-2 levels abrogated susceptibility to Par-4-dependent apoptosis, suggesting that Par-4-mediated apoptosis requires downmodulation of Bcl-2 levels. The inverse correlation between Par-4 and Bcl-2 expression was recapitulated in human prostate tumors. Par-4 but not Bcl-2 was detected in the secretory epithelium of benign prostatic tumors and in primary and metastatic prostate cancers that are apt to undergo apoptosis. Moreover, xenografts of human, androgen-dependent CWR22 tumors showed Par-4 but not Bcl-2 expression. By contrast, androgen-independent CWR22R tumors derived from the CWR22 xenografts showed mutually exclusive expression patterns of Par-4 and Bcl-2. These findings suggest a mechanism by which Par-4 may sensitize prostate tumor cells to apoptosis.

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.

The G1-phase growth-arresting action of interleukin-1 is independent of p53 and p21/WAF1 function.

Interleukin-1 (IL-1) causes G1-phase growth arrest of A375-C6 human melanoma cells by hypophosphorylation of the retinoblastoma susceptibility gene product Rb. Because p53 and p21/WAF1 proteins are key components of growth arrest pathways involving Rb hypophosphorylation, we tested the functional role of these two proteins in IL-1 action. Exposure to IL-1 caused induction of both p53 and p21/WAF1 proteins. However, inhibition of p53 function by the K1 mutant of SV40-T antigen or by m175 (Arg to His) dominant-negative mutant of p53 did not result in abrogation of IL-1 action, suggesting that p53 function is not required for growth arrest by IL-1. Studies aimed at testing the role of p21/WAF1 in IL-1 action indicated that IL-1 induced p21/WAF1 expression independently of the p53 status of the cells. However, inhibition of p21/WAF1 expression resulted in only a marginal rescue from the growth-arresting action of IL-1. These findings imply that despite their induction, neither wild-type p53 nor p21 can fully account for the growth arrest by IL-1. Thus, a p53- and p21-independent pathway(s) mediates IL-1 action.

Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer disease.

Prostate apoptosis response-4 (Par-4) is a protein containing both a leucine zipper and a death domain that was isolated by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis. Par-4 is expressed in the nervous system, where its function is unknown. In Alzheimer disease (AD), neurons may die by apoptosis, and amyloid beta-protein (A beta) may play a role in this. We report here that Par-4 expression is increased in vulnerable neurons in AD brain and is induced in cultured neurons undergoing apoptosis. Blockade of Par-4 expression or function prevented neuronal apoptosis induced by Ab and trophic factor withdrawal. Par-4 expression was enhanced, and mitochondrial dysfunction and apoptosis exacerbated, in cells expressing presenilin-1 mutations associated with early-onset inherited AD.