Diclofenac Enhances Docosahexaenoic Acid-Induced Apoptosis in Vitro in Lung Cancer Cells.

Polyunsaturated fatty acids (PUFAs) and non-steroidal anti-inflammatory drugs (NSAIDs) show anticancer activities through diverse molecular mechanisms. However, the anticancer capacities of either PUFAs or NSAIDs alone is limited. We examined whether combining NSAIDs with docosahexaenoic (DHA), commonly derived from fish oils, would possibly synergize their anticancer activity. We determined the viability of lung cancer cell lines (NCI-H1573, A549, NCI-H1299, and NCI-H1975) after exposure to DHA and various NSAIDs. We further conducted cell apoptosis assays and analyzed apoptosis-associated proteins and some key proteins in the RAS/MEK/ERK and PI3K/Akt pathways using western blot analysis. We also determined the impact of the treatment on the expression of inducible cancer-related genes using nCounter PanCancer Pathways gene expression analysis. The results showed that the combination of DHA and NSAIDs increased suppression of cell viability in all the lung cancer cell lines tested compared to each of the compounds used alone, with diclofenac being the most potent NSAID tested. This synergistic effect is especially significant in A549 and NCI-H1573 cells. The combination treatment was more effective at inhibiting clonogenic cell growth and anchorage-independent growth in soft agar, inducing caspase-dependent apoptosis, and altering expression of critical proteins in the RAS/MEK/ERK and PI3K/Akt pathways. The data from this study demonstrate that DHA combined with low dose diclofenac provides greater anticancer potential, which can be further developed for chemoprevention and adjunct therapy in lung cancer.

Polyisoprenylated Cysteinyl Amide Inhibitors Deplete K-Ras and Induce Caspase-dependent Apoptosis in Lung Cancer Cells.

BACKGROUND: Non-small cell lung cancers (NSCLC) harboring mutation-induced dysregulation of Ras signaling present some of the most difficult-to-manage cases, since directly targeting the constitutively active mutant Ras proteins has not resulted in clinically useful drugs. Therefore, modulating Ras activity for targeted treatment of cancer remains an urgent healthcare need. OBJECTIVE: In the current study, we investigated a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs), for their anticancer molecular mechanisms using the NSCLC cell panel with K-Ras and/or other mutant genes. METHODS: The effect of the PCAIs on intracellular K-Ras levels, cell viability, apoptosis, spheroid and colony formation were determined. RESULTS: Treatment of the lung cancer cells with the PCAIs, NSL-RD-035, NSL-BA-036, NSL-BA- 040 and NSL-BA-055 resulted in concentration-dependent cell death in both K-Ras mutant (A549, NCI-H460, and NCI-H1573), N-Ras mutant (NCI-H1299) and other (NCI-H661, NCI-H1975, NCIH1563) NSCLC cells. The PCAIs at 1.0 -10 µM induced the degeneration of 3D spheroid cultures, inhibited clonogenic cell growth and induced marked apoptosis via the extrinsic pathway. The most potent of the PCAIs, NSL-BA-055, at 5 µM induced a seven-fold increase in the activity of caspase- 3/7 and a 75% selective depletion of K-Ras protein levels relative to GAPDH in A549 cells that correlated with PCAIs-induced apoptosis. NSL-BA-040 and NSL-BA-055 also induced the phosphorylation of MAP kinase (ERK 1/2). CONCLUSION: Taken together, PCAIs may be potentially useful as targeted therapies that suppress NSCLC progression through disruption of Ras-mediated growth signaling.

Polyisoprenylated cysteinyl amide inhibitors induce caspase 3/7- and 8-mediated apoptosis and inhibit migration and invasion of metastatic prostate cancer cells.

Metastatic castration-resistant prostate cancer (mCRPC) is the most aggressive and deadly form of prostate cancer. It is characterized by the overexpression of epidermal growth factor receptors whose signals are mediated by small monomeric G proteins of the Ras superfamily. These require polyisoprenylation for functional activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were developed as potential targeted therapies to mitigate excessive growth signaling in mCRPC either by inhibiting PMPMEase and/or perturbing the polyisoprenylation-dependent functional interactions. We investigated the effects of PCAIs on the viability of prostate cancer PC 3, DU 145, MDA PCa 2b, LNCaP and 22Rv1 cells, determined the effect of the PCAIs on PC 3 cell proliferation, survival and caspase-mediated apoptotic cell death. Metastatic PC 3 and DU 145 cell migration and invasion in the presence of NSL-BA-040 were determined using the scratch and matrigel invasion assays. We further investigated the effect of NSL-BA-040 on F-actin organization in TagRFP F-actin marker-transfected metastatic PC 3 cells. The PCAIs suppress mCRPC cell viability with EC50 values ranging from 1.3 to 4.0 µM for the most potent of the PCAIs against PC 3, DU 145, MDA PCa 2b, LNCaP and 22Rv cells. PCAIs induced apoptotic cell death in PC 3 and DU 145 cells as determined by annexin V/propidium iodide flow cytometry analysis through the activation of caspases 3 and 8 while also inhibiting migration and invasion through the disruption of F-actin organization. Taken together, our studies show the anti-cancer effects on mCRPC cells through induction of caspase-mediated apoptosis and F-actin-mediated inhibition of cell motility and invasion thereby indicating the anti-tumor and anti-metastatic potential of the PCAIs.

Polyisoprenylated cysteinyl amide inhibitors disrupt actin cytoskeleton organization, induce cell rounding and block migration of non-small cell lung cancer.

The malignant potential of Non-Small Cell Lung Cancer (NSCLC) is dependent on cellular processes that promote metastasis. F-actin organization is central to cell migration, invasion, adhesion and angiogenesis, processes involved in metastasis. F-actin remodeling is enhanced by the overexpression and/or hyper-activation of some members of the Rho family of small GTPases. Therefore, agents that mitigate hyperactive Rho proteins may be relevant for controlling metastasis. We previously reported the role of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential inhibitors of cancers with hyperactive small GTPases. In this report, we investigate the potential role of PCAIs against NSCLC cells and show that as low as 0.5 µM PCAIs significantly inhibit 2D and 3D NCI-H1299 cell migration by 48% and 45%, respectively. PCAIs at 1 µM inhibited 2D and 3D NCI-H1299 cell invasion through Matrigel by 50% and 85%, respectively. Additionally, exposure to 5 µM of the PCAIs for 24 h caused at least a 66% drop in the levels of Rac1, Cdc42, and RhoA and a 38% drop in F-actin intensity at the cell membrane. This drop in F-actin was accompanied by a 73% reduction in the number of filopodia per cell. Interestingly, the polyisoprenyl group of the PCAIs is essential for these effects, as NSL-100, a non-farnesylated analog, does not elicit similar effects on F-actin assembly and organization. Our findings indicate that PCAIs disrupt F-actin assembly and organization to suppress cell motility and invasion. The PCAIs may be an effective therapy option for NSCLC metastasis and invasion control.

Disruption of actin filaments and suppression of pancreatic cancer cell viability and migration following treatment with polyisoprenylated cysteinyl amides.

Pancreatic cancer is characterized by K-Ras mutations in over 90% of the cases. The mutations make the tumors aggressive and resistant to current therapies resulting in very poor prognoses. Valiant efforts to drug mutant K-Ras and related proteins for the treatment of cancers with Ras mutations have been elusive. The need thus persists for therapies to target and suppress the hyperactive K-Ras mutant proteins to normal levels of activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were designed to disrupt polyisoprenylated protein metabolism and/or functions. The potential for PCAIs to serve as targeted anticancer agents for pancreatic cancer was evaluated in pancreatic ductal adenocarcinoma (PDAC) cell lines expressing mutant (MIAPaCa-2 and Panc-1) and wild type (BxPC-3) K-Ras proteins. The PCAIs inhibited MIAPaCa-2 and BxPC-3 cell viability and induced apoptosis with EC50 values as low as 1.9 µM. The PCAIs, at 0.5 µM, inhibited MIAPaCa-2 cell migration by 50%, inhibited colony formation and disrupted F-actin filament organization. The PCAIs blocked MIAPaCa-2 cell progression at the G0/G1 phase. These results reveal that the PCAIs disrupt pertinent biological processes that lead to pancreatic cancer progression and thus have the potential to act as targeted effective treatments for pancreatic cancer.

The antiangiogenic effects of polyisoprenylated cysteinyl amide inhibitors in HUVEC, chick embryo and zebrafish is dependent on the polyisoprenyl moiety.

Angiogenesis is essential for solid tumor growth, therapeutic resistance and metastasis, the latest accounting for 90% of cancer deaths. Although angiogenesis is essential for the malignant transformations in solid tumors and therefore is an attractive target, few drugs are available that block tumor angiogenesis. The focus has been to block signaling by receptor tyrosine kinases (RTKs), such as for vascular endothelial growth factor (VEGF), whose activation abrogate apoptosis and promote angiogenesis. The polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed to modulate aberrant polyisoprenylated small G-proteins such as mutant Ras whose constitutive activation promotes RTKs signaling. Since polyisoprenylation is essential for protein-protein interactions and functions of G-proteins, we hypothesized that the PCAIs would disrupt the monomeric G-protein signaling thereby effectively inhibiting angiogenesis. In this study we determined the effects of PCAIs on human umbilical vein endothelial cells (HUVEC) tube formation, cell viability, cell migration and invasion as well as in vivo using the chick chorioallantoic membrane (CAM) and zebrafish models. At sub- to low micromolar concentrations, the PCAIs inhibit the native and VEGF-stimulated cell migration and invasion as well as tube formation and angiogenesis in CAM and zebrafish embryos. The concentrations that block the angiogenic processes were lower than those that induce cell death. Since angiogenesis is essential for tumor growth but otherwise limited to wound healing, feeding fat cells and uterine wall repair in adults, it is conceivable that these compounds can be developed into safer therapeutics for cancers and retinal neovascularization that leads to loss of vision.

Polyisoprenylated methylated protein methyl esterase as a putative drug target for androgen-insensitive prostate cancer.

Prostate cancer (CaP) is the most frequently diagnosed cancer in US men, with an estimated 236,590 new cases and 29,720 deaths in 2013. There exists the need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant disease. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins have been implicated in CaP. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyses the only reversible and terminal reaction of the polyisoprenylation pathway and may promote the effects of G proteins on cell viability. In this review, the potential role of PMPMEase to serve as a new drug target for androgen-insensitive CaP was determined. Specific PMPMEase activities were found to be 3.5- and 4.5-fold higher in androgen-sensitive 22Rv1 and androgen-dependent LNCaP and 1.5- and 9.8-fold higher in castration-resistant DU 145 and PC-3 CaP cells compared to normal WPE1-NA22 prostate cells. The PMPMEase inhibitor, L-28, induced apoptosis with EC50 values ranging from 1.8 to 4.6 µM. The PMPMEase activity in the cells following treatment with L-28 followed a similar profile, with IC50 ranging from 2.3 to 130 µM. L-28 disrupted F-actin filament organisation at 5 µM and inhibited cell migration 4-fold at 2 µM. Analysis of a CaP tissue microarray for PMPMEase expression revealed intermediate, strong, and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to trace and weak staining in the normal and normal-adjacent tissue controls. The data are an indication that effective targeting of PMPMEase through the development of more potent agents may lead to the successful treatment of metastatic CaP.

Polyisoprenylated methylated protein methyl esterase: a putative biomarker and therapeutic target for pancreatic cancer.

Pancreatic cancer is the most deadly neoplasm with a 5-year survival rate of less than 6%. Over 90% of cases harbor K-Ras mutations, which are the most challenging to treat due to lack of effective therapies. Here, we reveal that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic ductal adenocarcinoma. We further present polyisoprenylated cysteinyl amide inhibitors (PCAIs) as novel compounds designed with structural elements for optimal in vivo activities and selective disruption of polyisoprenylation-mediated protein functions. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 µM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 µM while salirasib and farnesylthiosalicylamide were ineffective at 20 µM. The PCAIs thus have the potential to serve as effective therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins.

Polyisoprenylated methylated protein methyl esterase overexpression and hyperactivity promotes lung cancer progression.

The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 µM for A549 and 2.8 µM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer.

Polyisoprenylated methylated protein methyl esterase is both sensitive to curcumin and overexpressed in colorectal cancer: implications for chemoprevention and treatment.

Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with a K(i) of 0.3 µM (IC50 = 12.4 µM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yielded K M values of 23.6 ± 2.7 and 85.3 ± 15.3 µM in the absence or presence of 20 µM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 µg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50 of 22.6 µg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001). The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents.