Exploring CDKN1A Upregulation Mechanisms: Insights into Cell Cycle Arrest Induced by NC2603 Curcumin Analog in MCF-7 Breast Cancer Cells.

Breast cancer stands out as one of the most prevalent malignancies worldwide, necessitating a nuanced understanding of its molecular underpinnings for effective treatment. Hormone receptors in breast cancer cells substantially influence treatment strategies, dictating therapeutic approaches in clinical settings, serving as a guide for drug development, and aiming to enhance treatment specificity and efficacy. Natural compounds, such as curcumin, offer a diverse array of chemical structures with promising therapeutic potential. Despite curcumin's benefits, challenges like poor solubility and rapid metabolism have spurred the exploration of analogs. Here, we evaluated the efficacy of the curcumin analog NC2603 to induce cell cycle arrest in MCF-7 breast cancer cells and explored its molecular mechanisms. Our findings reveal potent inhibition of cell viability (IC50 = 5.6 µM) and greater specificity than doxorubicin toward MCF-7 vs. non-cancer HaCaT cells. Transcriptome analysis identified 12,055 modulated genes, most notably upregulation of GADD45A and downregulation of ESR1, implicating CDKN1A-mediated regulation of proliferation and cell cycle genes. We hypothesize that the curcumin analog by inducing GADD45A expression and repressing ESR1, triggers the expression of CDKN1A, which in turn downregulates the expression of many important genes of proliferation and the cell cycle. These insights advance our understanding of curcumin analogs' therapeutic potential, highlighting not just their role in treatment, but also the molecular pathways involved in their activity toward breast cancer cells.

The Transcriptome of BT-20 Breast Cancer Cells Exposed to Curcumin Analog NC2603 Reveals a Relationship between EGR3 Gene Modulation and Cell Migration Inhibition.

Breast cancer represents a critical global health issue, accounting for a substantial portion of cancer-related deaths worldwide. Metastasis, the spread of cancer cells to distant organs, is the primary cause of approximately 90% of breast cancer-related fatalities. Despite advances in cancer treatment, conventional chemotherapeutic drugs often encounter resistance and demonstrate limited efficacy against metastasis. Natural products have emerged as promising sources for innovative cancer therapies, with curcumin being one such example. However, despite its therapeutic potential, curcumin exhibits several limitations. Analogous compounds possessing enhanced bioavailability, potency, or specificity offer a promising avenue for overcoming these challenges and demonstrate potent anti-tumor activities. Our study investigates the antimetastatic potential of the curcumin analog NC2603 in breast cancer cells, utilizing BT-20 cells known for their migratory properties. Cell viability assessments were performed using the MTT reduction method, while migration inhibition was evaluated through scratch and Transwell migration assays. Transcriptome analysis via next-generation sequencing was employed to elucidate gene modulation and compound mechanisms, with subsequent validation using RT-qPCR. The IC50 of NC2603 was determined to be 3.5 µM, indicating potent inhibition of cell viability, and it exhibited greater specificity for BT-20 cells compared with non-cancerous HaCaT cells, surpassing the efficacy of doxorubicin. Notably, NC2603 demonstrated superior inhibition of cell migration in both scratch and Transwell assays compared with curcumin. Transcriptome analysis identified 10,620 modulated genes. We validated the expression of six: EGR3, ATF3, EMP1, SOCS3, ZFP36, and GADD45B, due to their association with migration inhibition properties. We hypothesize that the curcumin analog induces EGR3 expression, which subsequently triggers the expression of ATF3, EMP1, SOCS3, ZFP36, and GADD45B. In summary, this study significantly advances our comprehension of the intricate molecular pathways involved in cancer metastasis, while also examining the mechanisms of analog NC2603 and underscoring its considerable potential as a promising candidate for adjuvant therapy.

Two novel piperidones induce apoptosis and antiproliferative effects on human prostate and lymphoma cancer cell lines.

Cancer remains the second most common cause of death in the US. Due to a recurrent problem with anticancer drug resistance, there is a current need for anticancer drugs with distinct modes of action for combination drug therapy We have tested two novel piperidone compounds, named 2608 (1-dichloroacetyl - 3,5-bis(3,4-difluorobenzylidene)-4-piperidone) and 2610 (1-dichloroacetyl-3,5-bis(3,4-dichlorobenzylidene)-4-piperidone), for their potential cytotoxicity on numerous human cancer cell lines. We found that both compounds were cytotoxic for breast, pancreatic, leukemia, lymphoma, colon, and fibroblast cell lines, with a cytotoxic concentration 50% (CC50) in the low micromolar to nanomolar concentration range. Further assays focused primarily on an acute lymphoblastic lymphoma and colon cancer cell lines since they were the most sensitive and resistant to the experimental piperidones. The cell death mechanism was evaluated through assays commonly used to detect the induction of apoptosis. These assays revealed that both 2608 and 2610 induced reactive oxygen species (ROS) accumulation, mitochondrial depolarization, and activated caspase-3/7. Our findings suggest that the piperidones induced cell death via the intrinsic apoptotic pathway. Additional assays revealed that both piperidones cause cell cycle alteration in lymphoma and colon cell lines. Both piperidones elicited DNA fragmentation, as evidenced by an increment in the sub-G0/G1 subpopulation in both cell lines. Similar to other related compounds, both piperidones were found to act as proteasome inhibitors by increasing the levels of poly-ubiquitinated proteins in both lymphoma and colon cell lines. Hence, the two piperidones exhibited attractive cytotoxic properties and suitable mechanisms of action, which makes them good candidates as anticancer drugs.

Cytotoxic derivatives of dichloroacetic acid and some metal complexes.

This study outlines a number of studies of dichloroacetic acid (DCA) and some of its derivatives. Although DCA has low cytotoxic potencies, various structural modifications are described which result in potent cytotoxins. In particular, hybrid molecules created from DCA and other bioactive molecules whose modes of action differ from DCA are particularly promising as candidate anticancer agents. Considerable emphasis in this review is placed on various series of compounds that incorporate both platinum and DCA into their structures. In addition, the importance of the formulation of some of the bioactive compounds described herein is revealed.

Novel Unsymmetric 3,5-Bis(benzylidene)-4-piperidones That Display Tumor-Selective Toxicity.

Two series of novel unsymmetrical 3,5-bis(benzylidene)-4 piperidones 2a-f and 3a-e were designed as candidate antineoplastic agents. These compounds display potent cytotoxicity towards two colon cancers, as well as several oral squamous cell carcinomas. These compounds are less toxic to various non-malignant cells giving rise to large selectivity index (SI) figures. Many of the compounds are also cytotoxic towards CEM lymphoma and HL-60 leukemia cells. Representative compounds induced apoptotic cell death characterized by caspase-3 activation and subG1 accumulation in some OSCC cells, as well as the depolarization of the mitochondrial membrane potential in CEM cells. A further line of inquiry was directed to finding if the SI values are correlated with the atomic charges on the olefinic carbon atoms. The potential of these compounds as antineoplastic agents was enhanced by an ADME (absorption, distribution, metabolism, and excretion) evaluation of five lead molecules, which revealed no violations.

Design, Synthesis and Tumour-Selective Toxicity of Novel 1-[3-{3,5-Bis(benzylidene)-4-oxo-1-piperidino}-3-oxopropyl]-4-piperidone Oximes and Related Quaternary Ammonium Salts.

A novel series of 1-[3-{3,5-bis(benzylidene)-4-oxo-1-piperidino}-3-oxopropyl]-4-piperidone oximes 3a-h and related quaternary ammonium salts 4a-h were prepared as candidate antineoplastic agents. Evaluation against neoplastic Ca9-22, HSC-2 and HSC-4 cells revealed the compounds in series 3 and 4 to be potent cytotoxins with submicromolar CC50 values in virtually all cases. In contrast, the compounds were less cytocidal towards HGF, HPLF and HPC non-malignant cells revealing their tumour-selective toxicity. Quantitative structure-activity relationships revealed that, in general, both cytotoxic potency and selectivity index figures increased as the magnitude of the Hammett sigma values rose. In addition, 3a-h are cytotoxic towards a number of leukemic and colon cancer cells. 4b,c lowered the mitochondrial membrane potential in CEM cells, and 4d induced transient G2/M accumulation in Ca9-22 cells. Five compounds, namely 3c,d and 4c-e, were identified as lead molecules that have drug-like properties.

Novel hybrid molecules of 3,5-bis(benzylidene)-4-piperidones and dichloroacetic acid which demonstrate potent tumour-selective cytotoxicity.

A novel class of hybrid molecules 2a-o was designed as candidate antineoplastic agents from dichloroacetic acid which is a known inhibitor of pyruvate dehydrogenase kinase and a number of cytotoxic 3,5-bis(benzylidene)-4-piperidones 1. In general these new hybrid molecules are potent cytotoxins towards human HCT116 colon cancer cells. A number of lead molecules emerged having the IC50 values in the double digit nanomolar range. Most of these compounds are less toxic to human CRL1790 non-malignant colon cells and hence the selectivity index (SI) figures for most of the compounds are huge; in the case of 2c-g, m, n, the SI values are in excess of 100. Compounds 2g, 2j, 2m and 2n displayed >100-fold higher potency than the reference drug 5-FU. Quantitative structure-activity relationships revealed that the potencies of the compounds in series 2 increase as the magnitude of the Hammett σ and Taft σ* values rise. X-ray crystallographic of a representative compound 2c revealed various structural features which may influence cytotoxic potencies. Several representative compounds lowered the mitochondrial membrane potential and increased the production of reactive oxygen species in HCT116 cells. A minimal effect was noted in altering the percentage of cells in different phases of the cell cycle. Some futuredirections have been outlined for analog development.

3,5-Bis(3-dimethylaminomethyl-4-hydroxybenzylidene)-4-piperidone and related compounds induce glutathione oxidation and mitochondria-mediated cell death in HCT-116 colon cancer cells.

This study aims at investigating the cytotoxicity and some of the modes of action of 3,5-bis(3-dimethylamino-4-hydroxybenzylidene)-4-piperidone trihydrochloride 3 and two related compounds 2 (which lacks the dimethylaminomethyl groups) and 4 (which has an additional dimethylaminoethyl substituent in both aryl rings) in order to ascertain the contribution of dimethylaminoethyl substituent to bioactivity. The bioactivities of 2-4 were compared with curcumin 5. Both 2 and 3 displayed submicromolar GI50 values towards HCT-116 cells and were significantly more potent than 4, 5 and 5-fluorouracil (5-FU). All of the compounds displayed greater toxicity towards HCT-116 cells than human CRL-1790 non-malignant colon cells. In HCT-116 cells, the compounds 2, 3 and 5 increased the ratio of oxidised to reduced glutathione and destabilized the mitochondrial membrane potential. Both 2 and 5 produced an increase in mitochondrial superoxide and a burst in intracellular reactive oxygen species in HCT 116 cells. In addition, 2 and 4 stimulated respiration in rat liver mitochondria while 2 and 5 induced mitochondrial swelling. The results suggest that 2 and 5 cause oxidation or cross-linking of the thiols which control the mitochondrial permeability transition.

6-Benzylidene-2-[4-(pyridin-3-ylcarboxy)benzylidene]cyclohexanones: A novel cluster of tumour-selective cytotoxins.

Novel cytotoxins 3-5 containing the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore are disclosed. The compounds in series 3 and 5 have the potential to liberate niacin which may reduce some of the side effects of antineoplastic compounds. 3a-c emerged as the most potent cytotoxic compounds with IC50 values in the low micromolar range against human Molt4/C8 and CEM CD4+ T-lymphocytes as well as murine L1210 leukemia cells. QSAR studies revealed that cytotoxic potencies were negatively correlated with the magnitude of the Hammett sigma values of the aryl substituents. The compounds 3a-e displayed tumour-selective toxicity against human HL-60, HSC-2, HSC-3 and HSC-4 neoplasms as compared to human HGF, HPC and HPLF nonmalignant cells. A representative potent compound 3a caused PARP1 cleavage and G0/G1 cell cycle arrest in HSC-2 cells. These compounds are well tolerated in mice at doses up to and including 300mg/kg of the compounds and no mortalities were noted after 4h. The stability studies undertaken did not reveal that a representative compound 3a underwent hydrolysis to the related phenol 2a. Some guidelines for further analog development of the novel esters 3 were made.

1-[3-(2-Hydroxyethylsulfanyl)propanoyl]-3,5-bis(benzylidene)-4-piperidones: A novel cluster of P-glycoprotein dependent multidrug resistance modulators.

A series of 1-[3-(2-hydroxyethylsulfanyl)propanoyl]-3,5-bis(benzylidene)-4-piperidones 4a-e display promising P-glycoprotein dependent multidrug resistance (MDR) revertant properties and are significantly more potent than a reference drug verapamil when evaluated against L-5178Y MDR lymphoma cells. These dienones may be referred to as dual agents having both MDR revertant properties and tumour-selective cytotoxicity. In particular, 3,5-bis(4-chlorobenzylidene)-1-[3-(2-hydroxyethylsulfanyl]propanoyl-4-piperidone 4d emerged as a lead molecule for further development based on its MDR revertant properties, cytotoxic potencies and tumour-selective toxicity. The structure-activity relationships reveal important structural requirements for further designing of potent MDR revertants.

Tumour-specific cytotoxicity and structure-activity relationships of novel 1-[3-(2-methoxyethylthio)propionyl]-3,5-bis(benzylidene)-4-piperidones.

A series of 1-acyl-3,5-bis(benzylidene)-4-piperidones 3-7 were designed and synthesized as novel cytotoxic agents. These compounds displayed potent cytotoxic properties towards human Molt4/C8, CEM, HSC-2, HSC-3 and HSC-4 neoplasms and also to murine L1210 cells. The majority of the compounds have sub-micromolar or very low micromolar IC50 and CC50 values and are significantly more potent than the reference alkylating drug melphalan. Evaluation of these compounds against non-malignant HGF and HPLF cells revealed the tumour-specific toxicity. In particular, 3e emerged as a promising lead cytotoxic agent which caused apoptosis and PARP1 cleavage in HSC-2 cells.

Niacin esters of chalcones with tumor-selective properties.

Novel series of niacin esters of chalcones 2, 4 and 6 were designed as antineoplastic agents that have the potential to release the chemoprotectant niacin. These enones are cytotoxic to human CD4(+ )T-lymphocyte Molt 4/C8 and CEM and murine leukemia L1210 cells. Quantitative structure-activity relationship (QSAR) studies of the biodata in series 4 revealed that cytotoxic potency was enhanced by placing electron-repelling groups in one of the aryl rings. The compounds are lethal to HL-60, HSC-2, HSC-3 and HSC-4 neoplasms but less toxic to nonmalignant hepatocyte growth factor, hematopoietic progenitor cell and human periodontal ligament fibroblast cells. Hence, the compounds display tumor-selective toxicity. These chalcones are well tolerated in mice and no overt toxicity was noted. The results establish that in general the compounds in series 2, 4 and 6 have positive characteristics which warrant further studies.

Evaluation of α,ß-unsaturated ketones as antileishmanial agents.

In this study, we assessed the antileishmanial activity of 126 α,ß-unsaturated ketones. The compounds NC901, NC884, and NC2459 showed high leishmanicidal activity for both the extracellular (50% effective concentration [EC50], 456 nM, 1,122 nM, and 20 nM, respectively) and intracellular (EC50, 1,870 nM, 937 nM, and 625 nM, respectively) forms of Leishmania major propagated in macrophages, with little or no toxicity to mammalian cells. Bioluminescent imaging of parasite replication showed that all three compounds reduced the parasite burden in the murine model, with no apparent toxicity.

3-benzylidene-4-chromanones: a novel cluster of anti-tubercular agents.

In a quest for developing novel anti-tubercular agents, a series of 3-benzylidene-4-chromanones 1a-l were evaluated for growth inhibition of Mycobacterium tuberculosis H37Rv. Three promising compounds 1d, g, j emerged as the lead compounds with the IC50 and IC90 values of less than 1 µg/mL. Evaluation of the potent compounds 1d, g, j and k against Vero monkey kidney cells revealed that these compounds are far more toxic to M. tuberculosis than to Vero cells. Structure-activity relationships demonstrated that 3-benzylidene-4-chromanones are more potent against M. tuberculosis than the related 2-benzylidene cycloalkanones and the meta substituted chromanone derivatives are more active than their ortho- and para-counterparts. Some guidelines for amplifying the project are presented.

Cytotoxic 1,5-diaryl-3-oxo-1,5-pentadienes: an assessment and comparison of membrane permeability using Caco-2 and MDCK monolayers.

A number of cytotoxic conjugated unsaturated ketones were screened for their membrane permeability characteristics using Caco-2 and MDCK cells with the view of finding promising leads for in vivo evaluations. 3b-e and 4a-b demonstrated high permeability characteristics. In particular, 4a emerged as a promising lead which showed excellent apparent permeability (P(app): 54.70) and efflux ratio (ER: 0.15) values. In general, the relative apparent permeabilities of these enones are similar in both bioassays.

Regulation of N-myristoyltransferase by the calpain and caspase systems.

N-myristoyltransferase (NMT) is an essential eukaryotic enzyme which catalyzes the transfer of the myristoyl group to the terminal glycine residue of a number of proteins including those involved in signal transduction and apoptotic pathways. In higher eukaryotes, two isoforms of NMT have been identified (NMT1 and NMT2) which share about 76% amino acid sequence identity in humans. Protein-protein interactions of NMTs reveal that m-calpain interacts with NMT1 whereas caspase-3 interacts with NMT2. These findings reveal differential interactions of both isoforms of NMT with various signaling molecules. This minireview provides an overview of the regulation of N-myristoyltransferase by calpain and caspase systems.

Dimeric 3,5-Bis(benzylidene)-4-piperidones: Tumor-Selective Cytotoxicity and Structure-Activity Relationships.

Background: The objective of this study is to find novel antineoplastic agents that display greater toxicity to malignant cells than to neoplasms. In addition, the mechanisms of action of representative compounds are sought. This report describes the cytotoxicity of a number of dimers of 3,5-bis(benzylidene)-4-piperidones against human malignant cells (promyelocytic leukemia HL-60 and squamous cell carcinoma HSC-2, HSC-3, and HSC-4). Methods: Tumor specificity was evaluated by the selectivity index (SI), that is the ratio of the mean CC50 for human non-malignant oral cells (gingival fibroblasts, pulp cells, periodontal ligament fibroblasts) to that for malignant cells. Results: The compounds were highly toxic to human malignant cells. On the other hand, these molecules were less toxic to human non-malignant cells. In particular, a potent lead molecule, 3b, was identified. A QSAR study revealed that the placement of electron-releasing and hydrophilic substituents into the aryl rings led to increases in cytotoxic potencies. The modes of action of a lead compound discovered in this study designated 3b were the activation of caspases-3 and -7, as well as causing PARP1 cleavage and G2 arrest, followed by sub-G1 accumulation in the cell cycle. This compound also depolarized the mitochondrial membrane and generated reactive oxygen species in human colon carcinoma HCT116 cells. In conclusion, this study has revealed that, in general, the compounds described in this report are tumor-selective cytotoxins.

Mitochondrial dysfunction contributes to the cytotoxicity of some 3,5-bis(benzylidene)-4-piperidone derivatives in colon HCT-116 cells.

The objectives of this study are to investigate the possible ways by which the curcumin analogs 2a and 2b exert their antiproliferative properties. The analogs 2a and 2b have submicromolar IC(50) values towards human HCT-116 colon cancer cells but are far less toxic to human non-malignant CRL-1790 colon cells. Both compounds affected a number of mitochondrial functions in HCT-116 cells namely increasing the intracellular concentrations of reactive oxygen species, inhibiting oxygen consumption and decreasing the mitochondrial membrane potential. These molecules also produced swelling of isolated rat liver mitochondria, supporting a mitochondrial mechanism of cytotoxicity. Both compounds reacted with glutathione in the presence of glutathione S-transferase π and hence they may be classified as thiol alkylators.

From a cytotoxic agent to the discovery of a novel antimalarial agent.

A novel cytotoxin 3,5-bis(4-chlorobenzylidene)-1-[4-{2-(4-morpholinyl)ethoxy}phenyl-carbonyl]-4-piperidone hydrochloride 2 demonstrated potent antimalarial properties with IC(50) values of 0.60 and 1.97 µM against the drug sensitive D6 strain and the C235 drug-resistant strain of Plasmodium falciparum. This compound concentrates in red blood cells, lowers glutathione concentrations in erythrocytes and permeates across CACO-2 cells. These data reveal 2 to be a promising lead compound in the quest for novel antimalarial agents.

3,5-bis(benzylidene)-4-piperidones and related N-acyl analogs: a novel cluster of antimalarials targeting the liver stage of Plasmodium falciparum.

Drug resistance is a major challenge in antimalarial chemotherapy. In addition, a complete cure of malaria requires intervention at various stages in the development of the parasite within the host. There are only a few antimalarials that target the liver stage of the Plasmodium species which is an essential part of the life cycle of the malarial parasite. We report a series of antimalarial 3,5-bis(benzylidene)-4-piperidones and related N-acyl analogs 1-5, a number of which exhibit potent in vitro growth-inhibiting properties towards drug-sensitive D6 and drug-resistant C235 strains of Plasmodium falciparum as well as inhibiting the liver stage development of the malarial life cycle. The compounds 2b (IC50: 165 ng/mL), 3b (IC50: 186 ng/mL), 5c (IC50: 159 ng/mL) and 5d (IC50: 93.5 ng/mL) emerged as lead molecules that inhibit liver stage Plasmodium berghei and are significantly more potent than chloroquine (IC50: >2000 ng/mL) and mefloquine (IC50: >2000 ng/mL) in this screen. All the compounds that showed potent inhibitory activity against the P. berghei liver stage were nontoxic to human HepG2 liver cells (IC50: >2000 ng/mL). The compounds 5a and 5b exhibit comparable metabolic stability as chloroquine and mefloquine in human plasma and the most potent compound 5d demonstrated suitable permeability characteristics using the MDCK monolayer. These results emphasize the value of 3,5-bis(benzylidene)-4-piperidones as novel antimalarials for further drug development.