Pancreatic neuroendocrine tumors: Therapeutic challenges and research limitations.

Pancreatic neuroendocrine tumors (PNETs) are known to be the second most common epithelial malignancy of the pancreas. PNETs can be listed among the slowest growing as well as the fastest growing human cancers. The prevalence of PNETs is deceptively low; however, its incidence has significantly increased over the past decades. According to the American Cancer Society's estimate, about 4032 (> 7% of all pancreatic malignancies) individuals will be diagnosed with PNETs in 2020. PNETs often cause severe morbidity due to excessive secretion of hormones (such as serotonin) and/or overall tumor mass. Patients can live for many years (except for those patients with poorly differentiated G3 neuroendocrine tumors); thus, the prevalence of the tumors that is the number of patients actually dealing with the disease at any given time is fairly high because the survival is much longer than pancreatic ductal adenocarcinoma. Due to significant heterogeneity, the management of PNETs is very complex and remains an unmet clinical challenge. In terms of research studies, modest improvements have been made over the past decades in the identification of potential oncogenic drivers in order to enhance the quality of life and increase survival for this growing population of patients. Unfortunately, the majority of systematic therapies approved for the management of advanced stage PNETs lack objective response or at most result in modest benefits in survival. In this review, we aim to discuss the broad challenges associated with the management and the study of PNETs.

TW-37, a small-molecule inhibitor of Bcl-2, inhibits cell growth and induces apoptosis in pancreatic cancer: involvement of Notch-1 signaling pathway.

Overexpression of Bcl-2 family proteins has been found in a variety of aggressive human carcinomas, including pancreatic cancer, suggesting that specific agents targeting Bcl-2 family proteins would be valuable for pancreatic cancer therapy. We have previously reported that TW-37, a small-molecule inhibitor of Bcl-2 family proteins, inhibited cell growth and induced apoptosis in pancreatic cancer. However, the precise role and the molecular mechanism of action of TW-37 have not been fully elucidated. In our current study, we found that TW-37 induces cell growth inhibition and S-phase cell cycle arrest, with regulation of several important cell cycle-related genes like p27, p57, E2F-1, cdc25A, CDK4, cyclin A, cyclin D1, and cyclin E. The cell growth inhibition was accompanied by increased apoptosis with concomitant attenuation of Notch-1, Jagged-1, and its downstream genes such as Hes-1 in vitro and in vivo. We also found that down-regulation of Notch-1 by small interfering RNA or gamma-secretase inhibitors before TW-37 treatment resulted in enhanced cell growth inhibition and apoptosis. Our data suggest that the observed antitumor activity of TW-37 is mediated through a novel pathway involving inactivation of Notch-1 and Jagged-1.

Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway.

Despite rapid advances in many fronts, pancreatic cancer (PC) remains one of the most difficult human malignancies to treat due, in part, to de novo and acquired chemoresistance and radioresistance. Gemcitabine alone or in combination with other conventional therapeutics is the standard of care for the treatment of advanced PC without any significant improvement in the overall survival of patients diagnosed with this deadly disease. Previous studies have shown that PC cells that are gemcitabine-resistant (GR) acquired epithelial-mesenchymal transition (EMT) phenotype, which is reminiscent of "cancer stem-like cells"; however, the molecular mechanism that led to EMT phenotype has not been fully investigated. The present study shows that Notch-2 and its ligand, Jagged-1, are highly up-regulated in GR cells, which is consistent with the role of the Notch signaling pathway in the acquisition of EMT and cancer stem-like cell phenotype. We also found that the down-regulation of Notch signaling was associated with decreased invasive behavior of GR cells. Moreover, down-regulation of Notch signaling by siRNA approach led to partial reversal of the EMT phenotype, resulting in the mesenchymal-epithelial transition, which was associated with decreased expression of vimentin, ZEB1, Slug, Snail, and nuclear factor-kappaB. These results provide molecular evidence showing that the activation of Notch signaling is mechanistically linked with chemoresistance phenotype (EMT phenotype) of PC cells, suggesting that the inactivation of Notch signaling by novel strategies could be a potential targeted therapeutic approach for overcoming chemoresistance toward the prevention of tumor progression and/or treatment of metastatic PC.

SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status.

The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.

Critical role of prostate apoptosis response-4 in determining the sensitivity of pancreatic cancer cells to small-molecule inhibitor-induced apoptosis.

Role of prostate apoptosis response-4 (PAR-4) has been well described in prostate cancer. However, its significance in other cancers has not been fully elucidated. For the current study, we selected four pancreatic cancer cell lines (BxPC-3, Colo-357, L3.6pl, and HPAC) that showed differential endogenous expression of PAR-4. We found that nonpeptidic small-molecule inhibitors (SMI) of Bcl-2 family proteins (apogossypolone and TW-37; 250 nmol/L and 1 micromol/L, respectively) could induce PAR-4-dependent inhibition of cell growth and induction of apoptosis. Sensitivity to apoptosis was directly related to the expression levels of PAR-4 (R = 0.92 and R2 = 0.95). Conversely, small interfering RNA against PAR-4 blocked apoptosis, confirming that PAR-4 is a key player in the apoptotic process. PAR-4 nuclear localization is considered a prerequisite for cells to undergo apoptosis, and we found that the treatment of Colo-357 and L3.6pl cells with 250 nmol/L SMI leads to nuclear localization of PAR-4 as confirmed by 4',6-diamidino-2-phenylindole staining. In combination studies with gemcitabine, pretreatment with SMI leads to sensitization of Colo-357 cells to the growth-inhibitory and apoptotic action of a therapeutic drug, gemcitabine. In an in vivo setting, the maximum tolerated dose of TW-37 in xenograft of severe combined immunodeficient mice (40 mg/kg for three i.v. injections) led to significant tumor inhibition. Our results suggest that the observed antitumor activity of SMIs is mediated through a novel pathway involving induction of PAR-4. To our knowledge, this is the first study reporting SMI-mediated apoptosis involving PAR-4 in pancreatic cancer.

Chemoprevention of pancreatic cancer: characterization of Par-4 and its modulation by 3,3' diindolylmethane (DIM).

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or biological agents to suppress, reverse or prevent the carcinogenic process from turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique pro-apoptotic protein that selectively induces apoptosis in prostate cancer cells. However, its role in other malignancies has not been fully explored. This study tries to identify the functional significance of Par-4 in pancreatic cancer. METHODS: Multiple molecular techniques such as Western blot analysis, trypan blue assay for cell viability, MTT assay for cell growth inhibition and Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically its more bioavailable formulation, B-DIM, at low doses (20 micromol/L) induces Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM reduced cell viability and caused cell growth inhibition and apoptosis. Moreover, DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of Par-4 is indirectly related to increased sensitivity and cell death through apoptosis. To our knowledge the results reported here showed, for the first time, the induction of Par-4 by chemopreventive agents, in general, and DIM, in particular, in pancreatic cancer cells in vitro.

Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidative DNA breakage in normal cells at a concentration of 100-200 microM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 microM. would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol-Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties.

Resveratrol (3,4',5-trihydroxy stilbene), a plant derived polyphenol found in mulberries, grapes and red wine is considered to possess chemopreventive properties against cancer. It is recognized as a naturally occurring antioxidant but also catalyzes oxidative DNA degradation in vitro in the presence of transition metal ions such as copper. Using a cellular system of lymphocytes isolated from human peripheral blood and Comet assay, we have confirmed that resveratrol-Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. Also, trans-stilbene, which does not have any hydroxyl groups, is inactive in the lymphocyte system. Pre-incubation of lymphocytes with resveratrol indicates that it is capable of either traversing the cell membrane or binding to it. Our results are in partial support of our hypothesis that anticancer properties of various plant derived polyphenols may involve mobilization of endogenous copper and the consequent prooxidant action.