STAT3 induces anoikis resistance, promotes cell invasion and metastatic potential in pancreatic cancer cells.

Tumor cells need to attain anoikis resistance to survive prior to metastasis making it a vital trait of malignancy. The mechanism by which pancreatic cancer cells resist anoikis and metastasize is not well established. Significant proportion of pancreatic cancer cells resisted anoikis when grown under anchorage-independent conditions. The cells that resisted anoikis showed higher migratory and invasive characteristics than the cells that were cultured under anchorage-dependent condition. Interestingly, anoikis-resistant cells exhibited significantly increased expression and phosphorylation of signal transducer and activation of transcription 3 (STAT3) at Tyr 705, as compared to adherent cells. AG 490 and piplartine (PL) induced significant anoikis in anoikis-resistant pancreatic cancer cells. Silencing STAT3 not only reduced the capacity of pancreatic cancer cells to resist anoikis but also reversed its invasive characteristics. Interleukin-6 treatment and overexpression of STAT3 enhanced anoikis resistance and protected the cells from PL-induced anoikis. PL-treated cells completely failed to develop tumors when injected subcutaneously in immune-compromised mice. Moreover, these cells also failed to metastasize when injected intravenously. On the other hand, untreated anoikis-resistant cells not only formed aggressive tumors but also metastasized substantially to lungs and liver when injected intravenously. Metastatic nodules formed by untreated anoikis-resistant cells in lungs exhibited significant phosphorylation of STAT3 at Tyr705. Taken together, our results established the critical involvement of STAT3 in conferring anoikis resistance to pancreatic cancer cells and increased metastasis.

Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

BACKGROUND: Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood-brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer's disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model. RESULTS: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, "reduced-exposure" brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1. CONCLUSIONS: In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine content.

Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway.

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of AKT in the apoptosis-inducing effects of CAPE, cells were transfected with AKT. Our results revealed that AKT overexpression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, overexpression of XIAP further decreased CAPE-induced apoptosis. Taken together, our results suggest that CAPE suppresses phosphoinositide 3-kinase/AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo.

HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

Breast cancer metastasis is a multi-step process and requires cells to overcome anoikis. Anoikis is defined as cell-death that occurs due to loss of cell adhesion. During the course of cancer progression, tumor cells acquire resistance to anoikis. However, mechanisms of anoikis resistance are not clear. Human epidermal growth receptor 2 (HER2) overexpressing breast tumors are known to be highly aggressive and metastatic. The mechanisms correlating HER2 with metastasis are poorly understood. We observed increased anoikis resistance in HER2 overexpressing breast cancer cells. In addition, we identified that HER2 overexpression was also associated with increased sonic hedgehog (SHH) signaling especially GLI2, and that inhibition of SHH pathway suppressed anoikis resistance. GSK3ß is known to facilitate proteasome-mediated degradation of GLI2. Moreover, we observed that silencing of GLI2 resulted in reduced migration and invasion of HER2 overexpressing cells. Anoikis resistant HER2 overexpressing cells also showed increased rate and extent of metastasis in vivo, as compared to wild type anoikis resistant cells. Taken together, this study indicates a novel role of HER2/GSK3ß/GLI2 axis in anoikis resistance and metastasis, and that GLI2 could be a potential target for anti-cancer therapies.

Nanoemulsion formulations for anti-cancer agent piplartine--Characterization, toxicological, pharmacokinetics and efficacy studies.

Piplartine (PL) is an alkaloid found in black-pepper and known for its anticancer activity, however, due to poor solubility and lack of proper formulation, its use for oral administration is a challenge. The objective of this study was to formulate PL into nanoemulsion drug delivery system for oral delivery and thereafter evaluate toxicity, pharmacokinetics and therapeutic efficacy. Optimized nanoemulsions were formulated by self-emulsification as well as by homogenization-sonication method. Two nanoemulsions enhanced the solubility of PL with low polydispersity index and high stability. Both PL loaded nanoemulsions exhibited enhanced dissolution, cellular permeability and cytotoxic effects as compared to pure PL. Formulation of PL into nanoemulsions did not obstruct its cellular uptake in cancer cells. Blank or PL loaded nanoemulsions did not exhibited toxicity in mice upon daily oral administration for 60 days. Pharmacokinetics of PL followed a two-compartment model after intravenous administration. PL loaded nanoemulsions showed 1.5-fold increase in oral bioavailability as compared to free PL. Finally, PL loaded nanoemulsions showed marked anti-tumor activity at a dose of 10mg/kg in melanoma tumor bearing mice. In conclusion, for the first time we have developed a stable nanoemulsion delivery system for oral administration of PL, which enhanced its solubility, oral bioavailability and anti-tumor efficacy.

Modulation of signal transduction pathways by natural compounds in cancer.

Cancer is generally regarded as the result of abnormal growth of cells. According to World Health Organization, cancer is the leading cause of mortality worldwide. Mother nature provides a large source of bioactive compounds with excellent therapeutic efficacy. Numerous phytochemicals from nature have been investigated for anticancer properties. In this review article, we discuss several natural compounds, which have shown anti-cancer activity. Natural compounds induce cell cycle arrest, activate intrinsic and extrinsic apoptosis pathways, generate Reactive Oxygen Species (ROS), and down-regulate activated signaling pathways, resulting in inhibition of cell proliferation, progression and metastasis of cancer. Several preclinical studies have suggested that natural compounds can also increase the sensitivity of resistant cancers to available chemotherapy agents. Furthermore, combining FDA approved anti-cancer drugs with natural compounds results in improved efficacy. On the basis of these exciting outcomes of natural compounds against several cancer types, several agents have already advanced to clinical trials. In conclusion, preclinical results and clinical outcomes against cancer suggest promising anticancer efficacy of agents from natural sources.

Mechanisms of the Anticancer Effects of Isothiocyanates.

Cancer results from aberrant signaling pathways that result in uncontrolled cellular proliferation. The epidemiological studies have shown a strong inverse correlation between dietary consumption of cruciferous vegetables and incidences of cancer. Isothiocyanates (ITCs) are present in cruciferous vegetables like broccoli, cabbage, watercress, etc. and are identified as the major active constituents. Several mechanistic studies have demonstrated chemopreventive and chemotherapeutic activity of ITCs against various tumor types. ITCs exert anticancer activity by suppressing various critical hallmarks of cancer like cellular proliferation, angiogenesis, apoptosis, metastasis, etc., in vitro as well as in preclinical animal model. ITCs also generate reactive oxygen species to induce apoptosis in cancer cells. Due to promising preclinical results, few ITCs have also advanced to clinical trials. This chapter provides a candid review on the chemopreventive and chemotherapeutic activity of various major ITCs.

Inhibition of ß-catenin signaling suppresses pancreatic tumor growth by disrupting nuclear ß-catenin/TCF-1 complex: critical role of STAT-3.

Aberrant activation of ß-catenin/TCF signaling is related to the invasiveness of pancreatic cancer. In the present study, we evaluated the effect of capsaicin on ß-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of dishevelled (Dsh) protein DvI-1 in L3.6PL, PanC-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment induced GSK-3ß by inhibiting its phosphorylation and further activated APC and Axin multicomplex, leading to the proteasomal degradation of ß-catenin. Expression of TCF-1 and ß-catenin-responsive proteins, c-Myc and cyclin D1 also decreased in response to capsaicin treatment. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of ß-catenin. To establish the involvement of ß-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3ß. Our results reveal that capsaicin treatment suppressed LiCl or SB415286-mediated activation of ß-catenin signaling. Our results further showed that capsaicin blocked nuclear translocation of ß-catenin, TCF-1 and p-STAT-3 (Tyr705). The immunoprecipitation results indicated that capsaicin treatment reduced the interaction of ß-catenin and TCF-1 in the nucleus. Moreover, capsaicin treatment significantly decreased the phosphorylation of STAT-3 at Tyr705. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of ß-catenin and attenuated the effects of capsaicin in inhibiting ß-catenin signaling. Finally, capsaicin mediated inhibition of orthotopic tumor growth was associated with inhibition of ß-catenin/TCF-1 signaling. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with inhibition of ß-catenin signaling due to the dissociation of ß-catenin/TCF-1 complex and the process was orchestrated by STAT-3.

Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma.

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors.

Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. Here, we analyzed the role of STAT3 in anoikis resistance in melanoma cells leading to metastasis. When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions. The anoikis resistant cells also had significantly higher expression and phosphorylation of STAT3 at Y705 than the cells that were attached to the basement membrane. STAT3 inhibitors, AG 490 and piplartine (PL) induced anoikis in a concentration-dependent manner in anoikis resistant cells. Over-expression of STAT3 or treatment with IL-6 not only increased anoikis resistance, but also protected the cancer cells from PL-induced anoikis. On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate. STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized. In summary, our results for the first time establish STAT3 as a critical player that renders anoikis resistance to melanoma cells and enhance their metastatic potential.

Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb). Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length) and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.

CBP-mediated FOXO-1 acetylation inhibits pancreatic tumor growth by targeting SirT.

Here, we investigated the potential mechanism of capsaicin-mediated apoptosis in pancreatic cancer cells. Capsaicin treatment phosphorylated c-jun-NH2-kinase (JNK); forkhead box transcription factor, class O (FOXO1); and BIM in BxPC-3, AsPC-1, and L3.6PL cells. The expression of BIM increased in response to capsaicin treatment. Capsaicin treatment caused cleavage of caspase-3 and PARP, indicating apoptosis. Antioxidants tiron and PEG-catalase blocked capsaicin-mediated JNK/FOXO/BIM activation and protected the cells from apoptosis. Furthermore, capsaicin treatment caused a steady increase in the nuclear expression of FOXO-1, leading to increased DNA binding. Capsaicin-mediated expression of BIM was found to be directly dependent on the acetylation of FOXO-1. The expression of CREB-binding protein (CBP) was increased, whereas SirT-1 was reduced by capsaicin treatment. Using acetylation mimic or defective mutants, our result demonstrated that phosphorylation of FOXO-1 was mediated through acetylation by capsaicin treatment. JNK inhibitor attenuated the phosphorylation of FOXO-1, activation of BIM, and abrogated capsaicin-induced apoptosis. Moreover, silencing FOXO1 by siRNA blocked capsaicin-mediated activation of BIM and apoptosis, whereas overexpression of FOXO-1 augmented its effects. Silencing Bim drastically reduced capsaicin-mediated cleavage of caspase-3 and PARP, indicating the role of BIM in apoptosis. Oral administration of 5 mg/kg capsaicin substantially suppressed the growth of BxPC-3 tumor xenografts in athymic nude mice. Tumors from capsaicin-treated mice showed an increase in the phosphorylation of JNK, FOXO-1, BIM, and levels of CBP, cleavage of caspase-3, PARP, and decreased SirT-1 expression. Taken together, our results suggest that capsaicin activated JNK and FOXO-1, leading to the acetylation of FOXO-1 through CBP and SirT-1. Acetylated FOXO1 induced apoptosis in pancreatic cancer cells through BIM activation.