MicroRNA-375 and MicroRNA-221: Potential Noncoding RNAs Associated with Antiproliferative Activity of Benzyl Isothiocyanate in Pancreatic Cancer.

The deregulated presence or absence of microRNAs (miRNAs) might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. In this study, we provide evidence that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Interestingly, ectopic expression of miR-375 or the enforced silencing of miR-221 in cultured pancreatic cancer cells attenuates cell viability and sensitizes antiproliferative action of BITC. We also show that the expression of putative tumor suppressor miR-375 is more abundant in nonpathological mice pancreata than those with Kras(G12D)-driven pancreatic intraepithelial neoplasia (PanIN). To the contrary, the expression of oncogenic miR-221 is significantly elevated in the mouse pancreas with PanIN lesions. Although miR-375 has been shown to be aberrantly expressed in pancreatic cancer patients, there has not been a comprehensive study to investigate the molecular pathways targeted by this miRNA in pancreatic cancer cells. Further analysis by gene expression microarray revealed that IGFBP5 and CAV-1, potential biomarkers of pancreatic cancer, were significantly downregulated in cells transfected with miR-375. Correlatively, elevated expression of IGFBP5 and CAV-1 was evident in the mouse pancreas with preneoplastic lesions in which the expression of miR-375 wanes. Taken together, our findings suggest that anticancer agent BITC might target the expression of miR-221 and miR-375 to switch hyperproliferative pancreatic cancer cells to a hypoproliferative state.

Modulation of MicroRNAs by Chemical Carcinogens and Anticancer Drugs in Human Cancer: Potential Inkling to Therapeutic Advantage.

The disorder of microRNAs (miRNAs) often referred as 'micromanagers of gene expression' has been implicated with a vast array of neoplasmthe discovery establishes an important connection with the etiology, diagnosis and potential therapy of human cancer. Indeed, the wide range of profiling studies enabled to create miRNA signatures of solid tumors as well as cancers of blood origin. MiRNAs have been observed to play a significant role in the regulation of gene expression-a critical aspect of many biological processes, including cell development, differentiation, apoptosis and proliferation. The differential expression levels of miRNAs in tumors and their normal counterpart have enabled scientists to designate their roles as oncomir or tumor suppressor. Interestingly, the diminishment of oncogenic or enhanced levels of tumor suppressor miRNAs (antagomirs) have been reported to modulate the sensitivity of cancer cells to anticancer agents. To the other end, carcinogenic chemicals either possess the ability of silencing beneficial tumor suppressive miRNAs or maintain the augmented levels of their oncogenic counterpart. In this article we provide a comprehensive overview on the modulation of these "micromanaging oligos" by cancer causing as well as cancer preventing agents.

MicroRNA-mediated regulation of pancreatic cancer cell proliferation.

MicroRNAs (miRNAs) comprising 19-25 nucleotides are highly conserved small non-coding RNAs which regulate normal gene expression during development, cell proliferation and apoptosis by targeting mRNAs of protein-coding genes at the post-transcriptional level. Prevalent studies suggest that some human miRNAs, such as miRNA-16, are deregulated in human cancer and behave as tumor suppressors. The overall objective of our investigation was to assess whether miRNA-16 (miR-16) is involved in the regulation of critical genes, such as BCL2, that control the sensitivity of pancreatic cancer cells to apoptosis. This study showed that the ectopic overexpression of miR-16 may be therapeutically beneficial as is evidenced by impaired cell survival with concomitant attenuation of anti-apoptotic protein Bcl-2. Moreover, the luciferase reporter assay suggested that miR-16 post-transcriptionally regulates Bcl-2 expression in pancreatic cancer cells through the target sites of the 3' untranslated region of this gene.

Anti-proliferative and proapoptotic effects of benzyl isothiocyanate on human pancreatic cancer cells is linked to death receptor activation and RasGAP/Rac1 down-modulation.

Benzyl isothiocyanate can exert anti-tumor effect by arrest of cell cycle progression and induction of apoptosis in human pancreatic cancer cells. Among them, the dissection of the molecular mechanism of induction of apoptosis is important because the knowledge may be exploited for both cancer prevention and treatment. Our studies reported here indicate that BITC-mediated apoptosis involves the disappearance of intact 21-kDa Bid protein, cytochrome c release and predominant procaspase-3 cleavage. Using adenocarcinoma and metastatic pancreatic cancer cells, we investigated whether this dietary isothiocyanate induces apoptosis by converging two major pathways: the death receptor-mediated extrinsic and the mitochondrial intrinsic pathway. Indeed, cell surface receptor analysis by flow cytometry demonstrates the up-regulation of DR4, a member of death receptor family in BITC exposed pancreatic cancer cells. Since BITC is able to trigger death receptor signaling, we were interested in examining the effects of BITC and death receptor ligand TRAIL together on pancreatic cancer cell death. Interestingly, BITC augments TRAIL-induced apoptosis in both metastatic and adenocarcinoma cells. Moreover, we report for the first time that the sensitivity of metastatic pancreatic cancer cells to this isothiocyanate might be due to down-modulation of the proangiogenic molecule small GTPase Rac1 and caspase-3 substrate RasGAP, a regulator of Rho GTPase family.

2-Methoxyestradiol mediated signaling network in pancreatic cancer.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17 beta-estradiol, is known to be a potent inhibitor of neovascularization. Our previous studies have shown that 2-ME can suppress growth of pancreatic tumor cells in vitro and in vivo by the induction of apoptosis (Cancer Res 66: 4309-18, 2006). In order to better comprehend the signaling modulators of 2-ME in pancreatic cancer, we employed a PowerBlot Western Array screening system. Our proteomic profiling has provided framework to define the novel mechanisms of actions of 2-ME in pancreatic cancer. Interestingly, this high-throughput analysis identified proteins such as Rac1, Gelsolin, Glucocorticoid receptor (GR), Smad 2/3, Smad 4, IRS-1, which were not previously reported with 2-ME response. Interestingly, 2-ME modulated down regulation of GR level is accompanied by NF-k B activation in 2-ME responsive but not in resistant pancreatic cancer cells. In view of this observation, possible reciprocal relationship between GR and NF-kappaB activation might be an important regulatory factor in 2-ME mediated demise of a subpopulation of pancreatic cancer cells.

Combinatorial effect of epigallocatechin-3-gallate and TRAIL on pancreatic cancer cell death.

Epigallocatechin-3-gallate (EGCG), a major polyphenolic constituent of green tea, can exert growth suppressive effect on human pancreatic cancer cells by evoking apoptotic response. EGCG-induced apoptosis of pancreatic cancer cells is accompanied by growth arrest at an earlier phase of cell cycle along with depolarization of mitochondrial membrane. In this report, using MIA PaCa-2 cells as in vitro model, we demonstrate EGCG-induced cell death involves activation of caspase-8 and disappearance of intact 21 kDa Bid protein. Furthermore, exogenous expression of dominant negative caspase-8 or dominant negative FADD significantly abrogates apoptosis inducing ability of EGCG in MIA PaCa-2 cells. RNase protection assay revealed upregulation of the members of death receptor family, thus indicating the involvement of transmembrane extrinsic signaling in this polyphenol triggered pancreatic carcinoma cell death. Based on this, we examined the effect of EGCG and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) together on pancreatic cancer cells. A synergistic increase in apoptosis and cleavage of procaspase-3 was noted. Furthermore, clonogenic cell survival assay demonstrates the significant diminishment of MIA PaCa-2 cell proliferation in the presence of both EGCG and TRAIL. This combination treatment strategy has potential therapeutic advantage for pancreatic carcinoma.

Dietary isothiocyanate mediated apoptosis of human cancer cells is associated with Bcl-xL phosphorylation.

Benzylisothiocyanate (BITC), a major phase II enzyme inducer in the organic solvent of papaya fruit, has been shown to induce apoptosis specifically in cancer cells. The exposure of pancreatic, prostate as well as leukemic cells to this dietary isothiocyanate resulted in significant extent of apoptosis as evident from PARP cleavage, chromatin condensation or profound attenuation of procaspase-3 level. We also investigated whether BITC induces apoptosis by converging two major pathways: the death receptor mediated extrinsic and the mitochondrial intrinsic pathway. The exogenous expression of dominant-negative caspase-8 or dominant-negative caspase-9 can attenuate BITC-mediated cell death of prostate cancer cells. In parallel with this observation, BITC can activate both procaspase-8 and -9 in pancreatic and prostate cancer cells. Furthermore, flow cytometry analysis demonstrated the enrichment of sub-G0-G1 phase population with G2-M arrest in BITC challenged pancreatic cancer cells. In order to comprehend the molecular mechanism underlying the relationship between BITC-mediated cell cycle arrest and apoptosis we report here for the first time that the anti-apoptotic protein Bcl-xL was phosphorylated by BITC treatment. Subsequent investigation using Jun kinase inhibitor exhibits the involvement of Jun kinase in BITC triggered Bcl-xL phosphorylation and apoptosis.

Crosstalk between extrinsic and intrinsic cell death pathways in pancreatic cancer: synergistic action of estrogen metabolite and ligands of death receptor family.

2-Methoxyestradiol is a physiologic metabolite of 17beta-estradiol. This orally active compound can inhibit tumor growth or metastasis in tumor models without inducing any clinical sign of toxicity. Our previous studies indicated that 2-methoxyestradiol-mediated apoptosis involves the disappearance of intact 21-kDa Bid protein, cytochrome c release, and predominant procaspase-3 cleavage. Here, using MIA PaCa-2 cells as a model, we investigated whether this estrogen metabolite induces apoptosis by converging two major pathways: the death receptor-mediated extrinsic and the mitochondrial intrinsic pathway. Exogenous expression of dominant-negative caspase-8 or dominant-negative FADD reverts the effect of 2-methoxyestradiol-mediated cell death. In parallel with this observation, Z-IETD-FMK, a cell permeable irreversible inhibitor of caspase-8, can render significant protection against 2-methoxyestradiol-induced apoptosis. RNase protection assay and cell surface receptor analysis by flow cytometry show the up-regulation of members of death receptor family in 2-methoxyestradiol-exposed pancreatic cancer cells. Our mechanistic studies also implicate that oxidative stress precedes 2-methoxyestradiol-mediated c-Jun NH2-terminal kinase activation, leading to elevated Fas level. Because 2-methoxyestradiol is able to trigger death receptor signaling, we were interested in examining the effects of 2-methoxyestradiol and Fas ligand (FasL)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) together on pancreatic cancer cell death. Interestingly, the endogenous angiogenesis inhibitor 2-methoxyestradiol augments FasL/TRAIL-induced apoptosis in these cells. Moreover, the combination of 2-methoxyestradiol and TRAIL reduces the tumor burden in vivo in MIA PaCa-2 tumor xenograft model by caspase-3 activation.

Posttranslational modifications of Bcl2 family members--a potential therapeutic target for human malignancy.

Apoptosis is a process that can occur normally, such as during tissue remodeling, embryogenesis or abnormally during certain pathologies, such as cancer. The identification of the Bcl2 as well as IAP family members has suggested that excessive inhibition of apoptosis may constitute a common feature of all known human cancers-the ability to influence their onset, progression and outcome. Bcl2 family proteins are frequently regulated by phosphorylation that affects their activity and conformation. The structural analysis of antiapoptotic members of Bcl2 family has contributed to a better understanding of the functional domains including the discovery of an unstructured "loop region" (LR) near the N-terminus exposed to the cytoplasm. The antiapoptotic members of Bcl2 family such as Bcl2/Bcl-xL/Mcl-1 are phosphorylated on specific serine/threonine residues within this unstructured loop in response to diverse stimuli including treatment with chemotherapeutic taxanes, survival factor addition or chemopreventive agents. In most instances, such phosphorylation has been associated with the loss of their biological function. The chemoresistant tumors overexpress Bcl2/Bcl-xL/Mcl-1. To this end, the apoptosis yielding effect due to phosphorylation of antiapoptotic Bcl2 family members is quite interesting. Phosphorylation-dephosphorylation pathway of these antiapoptotic proteins should be an ideal molecular target for therapy of subpopulation of cancer in which these death repressors are essential prognostic markers. Thus, further gaining the knowledge on the mechanism of inactivation of Bcl2/Bcl-xL/Mcl-1 by phosphorylation might be of paramount importance to therapy for human malignancies in which overexpression of these antiapoptotic proteins plays an essential role.

Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells.

Polyphenols such as epigallocatechin-3-gallate (EGCG) from green tea extract can exert a growth-suppressive effect on human pancreatic cancer cells in vitro. In pursuit of our investigations to dissect the molecular mechanism of EGCG action on pancreatic cancer, we observed that the antiproliferative action of EGCG on pancreatic carcinoma is mediated through programmed cell death or apoptosis as evident from nuclear condensation, caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. EGCG-induced apoptosis of pancreatic cancer cells is accompanied by growth arrest at an earlier phase of the cell cycle. In addition, EGCG invokes Bax oligomerization and depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. EGCG-induced downregulation of IAP family member X chromosome linked inhibitor of apoptosis protein (XIAP) might be helpful to facilitate cytochrome c mediated downstream caspase activation. On the other end, EGCG elicited the production of intracellular reactive oxygen species (ROS), as well as the c-Jun N-terminal kinase (JNK) activation in pancreatic carcinoma cells. Interestingly, inhibitor of JNK signaling pathway as well as antioxidant N-acetyl-L-cysteine (NAC) blocked EGCG-induced apoptosis. To summarize, our studies suggest that EGCG induces stress signals by damaging mitochondria and ROS-mediated JNK activation in MIA PaCa-2 pancreatic carcinoma cells.

Restoration of silenced Peutz-Jeghers syndrome gene, LKB1, induces apoptosis in pancreatic carcinoma cells.

Germ line mutations of the LKB1 tumor suppressor gene lead to Peutz-Jeghers syndrome (PJS) with a predisposition to cancer. Previous reports suggest that inactivation of this tumor-suppressor gene plays a role in the pathogenesis of gastrointestinal hamartomas as well as several cancers, including adenocarcinoma of the pancreas. Here, we have shown that LKB1 gene is silenced in the pancreatic cancer cell line AsPC-1, but can be recovered by treatment with the methylation inhibitor, 5-aza-2'-deoxycytidine (5aza2dC). Restoring the level of LKB1 through gene transfer initiated mitochondria-mediated apoptosis in AsPC-1 cells, as evidenced by the release of cytochrome c from the mitochondria. By confocal microscopy as well as biochemical fractionation, we demonstrate that LKB1 is present in the nuclear and mitochondrial compartments of pancreatic cancer cells. Our observations also indicate that although functional p53 is absent, the p53 kin, p73, is inducible by doxorubicin in AsPC-1 cells. This suggests that LKB1-induced apoptosis is p53 independent but might be p73-mediated in the pancreatic tumor cell line, AsPC-1.

Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis.

Bcl-xL, a close homolog of Bcl2, is an important regulator of apoptosis and is overexpressed in human cancer. Phosphorylation of Bcl-xL can be induced by microtubule-damaging drugs such as taxol or 2-methoxyestradiol (2-ME). By site-directed mutagenesis studies, we have identified that serine 62 is the necessary site for taxol- or 2-ME-induced Bcl-xL phosphorylation in prostate cancer cells. Further studies with the inhibitor of Jun kinase (JNK) and phosphorylation null mutant of Bcl-xL reveal the augmentative role of JNK-mediated Bcl-xL phosphorylation in apoptosis of prostate cancer cells. In summary, our studies suggest that the phosphorylation of Bcl-xL by stress response kinase signaling might oppose the anti-apoptotic function of Bcl-xL to permit prostate cancer cells to die by apoptosis.

Signal-induced site specific phosphorylation targets Bcl2 to the proteasome pathway.

The oncogene derived protein Bcl2 and its family members such as Bcl-xL or Mcl-1 can confer negative control in the pathway of cellular suicide machinery. The reversible phosphorylation of the components in the apoptotic-signaling pathway is likely to be an important regulatory mechanism to control the fate of a cell. Previous reports by others and us demonstrate that phosphorylation of anti-apoptotic proteins such as Bcl2, Bcl-xL or Mcl-1 can regulate their function depending on the apoptotic trigger or cell type. Also, evidence is now accumulating that the ubiquitin proteasome pathway can play an important role in apoptosis. In order to understand whether any cross-talk exists between proteasome and Bcl2 phosphorylation pathways, studies were undertaken employing cell permeable proteasome inhibitors. When proteasomes were inactivated, enhanced accumulation of slower mobility forms of Bcl2 was clearly evident. Due to substitution of the major phosphorylation sites Ser 70, 87 to Ala, no such effect was observed. It is known that in contrary to phospho Bcl2, native Bcl2 (non-phosphoform) is unable to associate with cis-trans peptidyl prolyl isomerase Pin1-a key factor to regulate the fate of phosphoforms of Bcl2 and apoptosis. Thus the enhanced resistance to cell death exhibited by phosphorylation defective mutant Bcl2 might be attributed to its inability to associate with Pin1.

2-Methoxyestradiol induces mitochondria dependent apoptotic signaling in pancreatic cancer cells.

The antiproliferative action of 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite is specific for cancer cells and is mediated by the induction of programmed cell death or apoptosis. But the identity of the downstream effectors of apoptotic signaling induced by 2-ME is not known. In the present study, we explored the effect of 2-ME on apoptosis in a panel of human pancreatic cancer cell lines. We have identified two categories of pancreatic cancer cell lines, which are either sensitive to 2-ME such as MIA PaCa-2, CFPAC-1, PANC-1, or non-sensitive to 2-ME such as Hs 766T. The results presented here indicated that the cell lines responsive to 2-ME could undergo apoptosis either by G2-M arrest (PANC-1) with Bcl-x(L) phosphorylation or by the accumulation of tetraploid cells in G1-S region (MIA PaCa-2) without Bcl-2/ Bcl-x(L) phosphorylation. Furthermore, 2-ME induced apoptosis in pancreatic cancer cells is mitochondria dependent as evident by the release of cytochrome c into the cytosol. 2-ME exposed cells exhibit Bid cleavage that is accompanied by the translocation of Bax into the mitochondria. Also 2-ME could induce phosphorylation of Bcl-x(L) in G2-M arrested cells, thus indicating the involvement of various anti- and pro-apoptotic regulators in the signaling cascade. The dissection of differential response of pancreatic cancer cell lines holds promise for future therapeutic intervention.

Proteasomal degradation of human peptidyl prolyl isomerase pin1-pointing phospho Bcl2 toward dephosphorylation.

Microtubule inhibitor-induced Bcl2 phosphorylation is detrimental to its antiapoptotic function. Phosphorylation of Bcl2 predominantly occurs on two serine residues (70 and 87) in cells arrested at G2-M phase by microtubule disarraying agents. Phospho Bcl2 can associate with a cis-trans peptidyl prolyl isomerase, Pin1. Pin1 and its homologues are known to target the proline residue carboxyl terminal to the phosphorylated threonine or serine residue of mitotic phosphoproteins, such as Bcl2. However, it was not clear how an extranuclear protein could associate with nuclear Pin1. The confocal images of the immunofluorescence studies employing phospho Bcl2-specific antibody developed in the laboratory demonstrated the translocation of phospho Bcl2 inside the nucleus. Interestingly, proteasomal degradation of Pin1 facilitates dephosphorylation of phospho Bcl2 due to longer exposure of Taxol. Here we show for the first time that proteasomal degradation of Pin1 is the key factor to determine the fate of phosphoforms of Bcl2. When Pin1 is degraded by proteasomes, phospho Bcl2 is converted to its native form. Thus, transient conformational change of Bcl2 due to association with peptidyl prolyl isomerase can contribute to irreversible apoptotic signaling.

Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KChAP/PIAS3beta in prostate cancer cells.

K(+) channel-associated protein/protein inhibitor of activated STAT (KChAP/PIAS3beta) is a potassium (K(+)) channel modulatory protein that boosts protein expression of a subset of K(+) channels and increases currents without affecting gating. Since increased K(+) efflux is an early event in apoptosis, we speculated that KChAP might induce apoptosis through its up-regulation of K(+) channel expression. KChAP belongs to the protein inhibitor of activated STAT family, members of which also interact with a variety of transcription factors including the proapoptotic protein, p53. Here we report that KChAP induces apoptosis in the prostate cancer cell line, LNCaP, which expresses both K(+) currents and wild-type p53. Infection with a recombinant adenovirus encoding KChAP (Ad/KChAP) increases K(+) efflux and reduces cell size as expected for an apoptotic volume decrease. The apoptosis inducer, staurosporine, increases endogenous KChAP levels, and LNCaP cells, 2 days after Ad/KChAP infection, show increased sensitivity to staurosporine. KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15. Consistent with activation of p53 as a transcription factor, p21 levels are increased in infected cells. Wild-type p53 is not essential for induction of apoptosis by KChAP, however, since KChAP also induces apoptosis in DU145 cells, a prostate cancer cell line with mutant p53. Consistent with its proapoptotic properties, KChAP prevents growth of DU145 and LNCaP tumor xenografts in nude mice, indicating that infection with Ad/KChAP might represent a novel method of cancer treatment.