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1.
Curr Med Chem ; 28(18): 3476-3489, 2021.
Article in English | MEDLINE | ID: mdl-33176634

ABSTRACT

Aberrant activation of monomeric G-protein signaling pathways drives some of the most aggressive cancers. Suppressing these hyperactivities has been the focus of efforts to obtain targeted therapies. Polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in various cancers. Its inhibition induces the death of cancer cells that harbor the constitutively active K-Ras proteins. Furthermore, the viability of cancer cells driven by factors upstream of K-Ras, such as overexpressed growth factors and their receptors or the mutationally-activated receptors, is also susceptible to PMPMEase inhibition. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were thus designed to target cancers with hyperactive signaling pathways involving the G-proteins. The PCAIs were, however, poor inhibitors of PMPMEase, with Ki values ranging from 3.7 to 20 µM. On the other hand, they inhibited cell viability, proliferation, colony formation, induced apoptosis in cells with mutant K-Ras and inhibited cell migration and invasion with EC50 values of 1 to 3 µM. HUVEC tube formation was inhibited at submicromolar concentrations through their disruption of actin filament organization. At the molecular level, the PCAIs at 2 to 5 µM depleted monomeric G-proteins such as K-Ras, RhoA, Cdc42 and Rac1. The PCAIs also deplete vinculin and fascin that are involved in actin organization and function while disrupting vinculin punctates in the process. These demonstrate a polyisoprenylation-dependent mechanism that explains the observed PCAIs' inhibition of the proliferative, invasive and angiogenic processes that promote both tumor growth and metastasis.


Subject(s)
Amides , Neoplasms , Amides/pharmacology , Cell Movement , Cell Survival , Humans , Neoplasms/drug therapy , Signal Transduction
2.
ACS Omega ; 5(51): 32907-32918, 2020 Dec 29.
Article in English | MEDLINE | ID: mdl-33403252

ABSTRACT

Triple-negative breast cancer (TNBC) is one of the most malignant cancers associated with early metastasis, poor clinical prognosis, and high recurrence rate. TNBC is a distinct subtype of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptors (HER2). Development of effective TNBC therapies has been limited partially due to the lack of specific molecular targets and chemotherapy involving different cytotoxic drugs suffers from significant side effects and drug-resistance development. Therefore, there is an unmet need for the development of novel and efficient therapeutic drugs with reduced side effects to treat TNBC. We have previously reported that certain analogues of haloperidol (a typical antipsychotic drug used for treating mental/mood disorders such as schizophrenia and bipolar disorder) suppress the viability of a variety of solid tumor cell lines, and we have identified 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluoro-phenyl)butan-1-one (SYA013) with such antiproliferative properties. Interestingly, unlike haloperidol, SYA013 shows moderate selectivity toward σ2 receptors. In this study, we explored the potential of SYA013 in modulating the important biological events associated with cell survival and progression as well as the mechanistic aspects of apoptosis in a representative TNBC cell line (MDA-MB-231). Our results indicate that SYA013 inhibits the proliferation of MDA-MB-231 cells in a concentration-dependent manner and suppresses cell migration and invasion. Apoptotic studies were also conducted in MDA-MB-468 cells (cells derived from a 51-year old Black female with metastatic adenocarcinoma of the breast.). In addition, we have demonstrated that SYA013 induces MDA-MB-231 cell death through the intrinsic apoptotic pathway and may suppress tumor progression and metastasis. Taken together, our study presents a mechanistic pathway of the anticancer properties of SYA013 against TNBC cell lines and suggests a potential for exploring SYA013 as a lead agent for development against TNBC.

3.
Curr Cancer Drug Targets ; 19(10): 838-851, 2019.
Article in English | MEDLINE | ID: mdl-30914025

ABSTRACT

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.


Subject(s)
Amides/pharmacology , Apoptosis/drug effects , Cell Proliferation , Lung Neoplasms/pathology , Mutation , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Spheroids, Cellular
4.
Anticancer Res ; 38(11): 6091-6098, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30396923

ABSTRACT

BACKGROUND/AIM: Coumarins remain one of the most versatile classes of compounds for anticancer drug design and discovery. The present study aimed to evaluate the in vitro cytotoxic activity of 7,8-Dihydroxy-3-arylcoumarin derivatives (7a-i) in A549, MDA-MB-231and PC-3 cancer cell lines. MATERIALS AND METHODS: Cell viability, cell-cycle progression and regulatory protein expression were evaluated using crystal violet dye-binding assay, flow cytometry and western blot analysis. RESULTS: 7,8-Diacetoxy-3-(4-nitrophenyl)coumarin (7h) showed the highest cytotoxic activity with CC50 of 7.51±0.07 µM in MDA-MB-231 cell line. The mechanism of cytotoxic action indicated that 7h caused significant (p<0.05) MDA-MB-231 cells arrest in the S phase as well as moderate cells arrest in the G2/M phase; confirmed by up-regulation of cyclins A/B1, p21 and CDKs 4/6, and down-regulation of cyclin E2 and CDK2 regulatory proteins. CONCLUSION: These results suggest that 7h could serve as a valuable template for the development of novel synthetic compounds for breast cancer treatment.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle Checkpoints/drug effects , Coumarins/pharmacology , S Phase/drug effects , A549 Cells , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Female , Humans
5.
Oncotarget ; 9(40): 25781-25795, 2018 May 25.
Article in English | MEDLINE | ID: mdl-29899821

ABSTRACT

Migratory cells form extracellular matrix attachments called focal-adhesions. Focal adhesion assembly and disassembly are regulated by the Rho family of small GTPases. We previously reported that polyisoprenylated cysteinyl amide inhibitors (PCAIs) suppress Rho protein levels, disrupting F-actin cytoskeleton remodeling in the formation of lamellipodia and filopodia. In this study, we investigated whether these observations effect focal adhesion formation, which involves cell surface receptors known as integrins and several signaling/adaptor proteins such as vinculin, α-actinin, Rock kinases and phospho-Myosin Light Chain-2 (p-MLC-2), that foster the linkage of the actin cytoskeleton to the extracellular matrix. We observed that treatment of H1299 cells with 5 µM PCAIs for 24 h markedly diminished the level of full-length integrin α4 by at least 24% relative to controls. PCAIs at 5 µM, diminished the levels of vinculin by at least 50%. Immunofluorescent analysis showed at least a 76% decrease in the number of vinculin-focal adhesion punctates. In addition, PCAIs diminished Rock1 levels by 25% and its substrate, p-MLC-2 by 75%. PCAIs did not significantly alter the levels of integrin ß5, α-actinin, and Rock2, suggesting that the effects of the PCAIs are target specific. Our data indicate that the PCAIs alter the levels of the Rho proteins and their effectors to abrogate their functions in cytoskeleton remodeling thereby suppressing focal adhesion formation. This in turn results in a PCAIs-induced decrease in cell invasion, thus making the PCAIs propitious agents for the inhibition of cancer growth and metastasis.

6.
Am J Cancer Res ; 7(7): 1515-1527, 2017.
Article in English | MEDLINE | ID: mdl-28744401

ABSTRACT

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.

7.
Oncotarget ; 8(19): 31726-31744, 2017 May 09.
Article in English | MEDLINE | ID: mdl-28423648

ABSTRACT

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.


Subject(s)
Actins/metabolism , Amides/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Movement/drug effects , Lung Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Humans , Protein Binding , Pseudopodia/drug effects , Pseudopodia/metabolism , Spheroids, Cellular , Tumor Cells, Cultured , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism
8.
Am J Cancer Res ; 6(11): 2532-2546, 2016.
Article in English | MEDLINE | ID: mdl-27904769

ABSTRACT

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.

9.
J Mol Biol ; 428(24 Pt B): 4929-4945, 2016 12 04.
Article in English | MEDLINE | ID: mdl-27760305

ABSTRACT

Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gαs protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.


Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Prenylation , Protein Processing, Post-Translational , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/metabolism , Amino Acid Sequence , Cell Line , Humans , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Conformation , Protein Interaction Mapping , Sequence Alignment , rap GTP-Binding Proteins/chemistry , rap1 GTP-Binding Proteins/chemistry
10.
Oncotarget ; 7(42): 68194-68205, 2016 Oct 18.
Article in English | MEDLINE | ID: mdl-27626690

ABSTRACT

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.


Subject(s)
Amides/pharmacology , Angiogenesis Inhibitors/pharmacology , Chorioallantoic Membrane/drug effects , Embryo, Nonmammalian/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Neovascularization, Physiologic/drug effects , Amides/chemistry , Animals , Butadienes/chemistry , Cell Movement/drug effects , Cell Survival/drug effects , Cells, Cultured , Chick Embryo , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/embryology , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/embryology , Hemiterpenes/chemistry , Human Umbilical Vein Endothelial Cells/physiology , Humans , Pentanes/chemistry , Polymers/chemistry , Zebrafish
11.
Sci Signal ; 6(277): ra39, 2013 May 28.
Article in English | MEDLINE | ID: mdl-23716716

ABSTRACT

During metastasis, cancer cells acquire the ability to dissociate from each other and migrate, which is recapitulated in vitro as cell scattering. The small guanosine triphosphatase (GTPase) Rap1 opposes cell scattering by promoting cell-cell adhesion, a function that requires its prenylation, or posttranslational modification with a carboxyl-terminal isoprenoid moiety, to enable its localization at cell membranes. Thus, signaling cascades that regulate the prenylation of Rap1 offer a mechanism to control the membrane localization of Rap1. We identified a signaling cascade initiated by adenosine A2B receptors that suppressed the prenylation of Rap1B through phosphorylation of Rap1B, which decreased its interaction with the chaperone protein SmgGDS (small GTPase guanosine diphosphate dissociation stimulator). These events promoted the cytosolic and nuclear accumulation of nonprenylated Rap1B and diminished cell-cell adhesion, resulting in cell scattering. We found that nonprenylated Rap1 was more abundant in mammary tumors than in normal mammary tissue in rats and that activation of adenosine receptors delayed Rap1B prenylation in breast, lung, and pancreatic cancer cell lines. Our findings support a model in which high concentrations of extracellular adenosine, such as those that arise in the tumor microenvironment, can chronically activate A2B receptors to suppress Rap1B prenylation and signaling at the cell membrane, resulting in reduced cell-cell contact and promoting cell scattering. Inhibiting A2B receptors may be an effective method to prevent metastasis.


Subject(s)
Adenosine/metabolism , Cell Movement/physiology , Models, Biological , Neoplasm Metastasis/physiopathology , Signal Transduction/physiology , Tumor Microenvironment , rap GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Immunoblotting , Immunoprecipitation , Microscopy, Confocal , Molecular Sequence Data , Prenylation , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2B/metabolism , rap GTP-Binding Proteins/genetics
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