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.

Differential protection by anthocyanin-rich bilberry extract and resveratrol against lipid micelle-induced oxidative stress and monolayer permeability in Caco-2 intestinal epithelial cells.

Excess dietary fat, and associated bile acids, can impair intestinal barrier integrity, produce intestinal or systemic inflammation and promote tumorigenesis. Dietary polyphenols in foods such as berries display antioxidant and other protective effects in many biological systems, but little is known about their protective effects on intestinal epithelial cells exposed to dietary fat. In a Caco-2 cell model of dietary fat-induced intestinal epithelial cell cytotoxicity, oxidative stress and barrier impairment, we investigated the relative protection afforded by an anthocyanin-rich bilberry extract (ARBE) or resveratrol. Exposure of the cells to mixed micelles (MM) of fatty acids and bile acids for 24 h markedly increased intracellular reactive oxygen species (ROS) and mitochondrial superoxide generation, decreased cell viability, increased expression of TNF-α mRNA and disrupted differentiated monolayer integrity. Starting prior to exposure to MM, treatments with ARBE or resveratrol, at polyphenol concentrations from 1.25-20 µM, strongly attenuated MM-induced intracellular ROS generation, and ARBE but not resveratrol decreased mitochondrial superoxide generation. Both ARBE and resveratrol inhibited the MM-induced expression of TNF-α mRNA. In assessments of differentiated monolayer integrity by transepithelial electrical resistance (TEER) and paracellular permeability, resveratrol protection was apparent at 3 h of MM exposure, but less at 6 h and absent by 9 h. In contrast, ARBE largely reversed MM-induced impairment by 9 h, with TEER values reaching 82% of control and the MM-induced paracellular permeability reduced by 78%. While they appeared to act differently, the results suggest that dietary sources of anthocyanins and resveratrol can help confer resistance of intestinal epithelial cells to oxidative stress and inflammation, and resultant barrier dysfunction and tumorigenesis, induced by high dietary fat common in the Western diet.

Doxorubicin Cytotoxicity in Differentiated H9c2 Cardiomyocytes: Evidence for Acute Mitochondrial Superoxide Generation.

Although a mitochondrial redox-cycling superoxide-generating mechanism for the cardiotoxicity of doxorubicin was suggested from experiments with isolated mitochondria, its occurrence and contribution to cytotoxicity in intact cardiomyocytes is not fully established. Therefore, we determined the immediate and delayed effects of doxorubicin on the generation of reactive oxygen species (ROS) and cytotoxicity in differentiated H9c2 cardiomyocytes. Although relatively short incubations (3 or 6 h) with 1 or 5 µM doxorubicin did not acutely decrease cell survival, exposure to 5 µM doxorubicin for 3 h was sufficient to cause a significant delayed decrease in cell survival after an additional 24 h without doxorubicin. Mitochondrial superoxide generation was observed to increase within 30 min of incubation with 5 µM doxorubicin. Increased intracellular ROS generation, decreased mitochondrial metabolic activity, and decreased mitochondrial membrane potential (MMP) were observed after more extended periods (6-12 h). Overall, these observations support that the toxicity of doxorubicin to differentiated cardiomyocytes involves acute mitochondrial superoxide generation with subsequent intracellular ROS generation, mitochondrial dysfunction, and cell death.

Increased mitochondrial content and function by resveratrol and select flavonoids protects against benzo[a]pyrene-induced bioenergetic dysfunction and ROS generation in a cell model of neoplastic transformation.

Dietary polyphenols act in cancer prevention and may inhibit carcinogenesis. A possible mitochondrial mechanism for carcinogen-induced neoplastic transformation and chemoprevention by polyphenols, however, is largely unexplored. Using the Bhas 42 cell model of carcinogen-induced neoplastic transformation, we investigated benzo[a]pyrene (B[a]P) along with different polyphenols for their effects on mitochondrial content and function, and on mitochondrial and intracellular ROS generation. Bhas 42 cells were either co-treated with 5 µM polyphenol starting 2 h before exposure to 4 µM B[a]P for 24 or 72 h, or pre-treated with polyphenol for 24 h and removed prior to B[a]P exposure. Exposure to B[a]P decreased mitochondrial content (by 46% after 24 h and 30% after 72 h), decreased mitochondrial membrane potential and cellular ATP, and increased generation of mitochondrial superoxide and intracellular ROS. Polyphenol co-treatments protected against the decreased mitochondrial content, with resveratrol being the most effective (increasing the mitochondrial content after 72 h by 75%). Measurements after 24 h of mRNA for mitochondria-related proteins and of SIRT1 enzyme activity suggested an involvement of increased mitochondrial biogenesis in the polyphenol effects. The polyphenol co-treatments also ameliorated B[a]P-induced deficits in mitochondrial function (most strongly resveratrol), and increases in generation of mitochondrial superoxide and intracellular ROS. Notably, 24 h pre-treatments with polyphenols strongly suppressed subsequent B[a]P-induced increases, after 24 and 72 h, in mitochondrial superoxide and intracellular ROS generation, with resveratrol being the most effective. In conclusion, the results support a mechanism for B[a]P carcinogenesis involving impaired mitochondrial function and increased mitochondria-derived ROS, that can be ameliorated by dietary polyphenols. The evidence supports an increase in mitochondrial biogenesis behind the strong chemoprevention by resveratrol, and a mitochondrial antioxidant effect in chemoprevention by quercetin.

Phenolic Breakdown Products of Cyanidin and Quercetin Contribute to Protection against Mitochondrial Impairment and Reactive Oxygen Species Generation in an In Vitro Model of Hepatocyte Steatosis.

A question in cell culture and dietary studies on protection by flavonoids against conditions such as hepatocyte steatosis is whether effects might be due to phenolic breakdown/digestion products. In HepG2 hepatocytes, treatment with quercetin, cyanidin, or their phenolic breakdown/digestion products (protocatechuic acid, 2,4,6-trihydroxybenzaldehyde, and caffeic acid), starting 2 h prior to oleic acid for 24 h, protected similarly against increases in intracellular lipid and reactive oxygen species and decreased mitochondrial membrane potential. Cyanidin or the phenolic products also protected against decreased mitochondrial content. After preincubation for only 1 h (to limit spontaneous degradation) and removal prior to oleic acid, only the phenolic products protected against decreased mitochondrial content, and without adding oleic acid, only protocatechuic acid and caffeic acid, and less so cyanidin, induced mitochondrial content. The results suggest that phenolic breakdown/digestion products of cyanidin and quercetin contribute to the protective effects in vitro, and perhaps in vivo.

Dietary Polyphenols Protect Against Oleic Acid-Induced Steatosis in an in Vitro Model of NAFLD by Modulating Lipid Metabolism and Improving Mitochondrial Function.

In this study, we aimed to determine the relative effectiveness of common dietary polyphenols or the isoquinoline alkaloid berberine in protecting against molecular mechanisms underlying non-alcoholic fatty liver disease (NAFLD) involving changes to cellular lipid metabolism and bioenergetics. In a model of steatosis using HepG2 hepatocytes, exposure of the cells to 1.5 mM oleic acid (OA) for 24 h caused steatosis and distorted cell morphology, induced the expression of mRNA for enzymes that are involved in lipogenesis and fatty acid oxidation (FAS and CPT1A), and impaired indices of aerobic energy metabolism (PPARγ mRNA expression, mitochondrial membrane potential (MMP), and galactose-supported ATP production). Co-treatment with 10 µM of selected polyphenols all strongly protected against the steatosis and changes in cell morphology. All polyphenols, except cyanidin, inhibited the effects on FAS and PPARγ and further increased CPT1A1 expression, suggesting a shift toward increased ß-oxidation. Resveratrol, quercetin, catechin, and cyanidin, however not kuromanin or berberine, ameliorated the decreases in MMP and galactose-derived ATP. Berberine was unique in worsening the decrease in galactose-derived ATP. In further investigations of the mechanisms involved, resveratrol, catechin, and berberine increased SIRT1 enzyme activity and p-AMPKαThr172 protein, which are involved in mitochondrial biogenesis. In conclusion, selected polyphenols all protected against steatosis with similar effectiveness, however through different mechanisms that increased aerobic lipid metabolism and mitochondrial function.

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.

Polyphenol inhibition of benzo[a]pyrene-induced oxidative stress and neoplastic transformation in an in vitro model of carcinogenesis.

While dietary polyphenols are widely recognized for cancer-preventing characteristics, the relative effectiveness and mechanisms of action of different polyphenols is not clear. In the present study, we investigated the protective effects of six different polyphenols against benzo[a]pyrene (B[a]P)-induced oxidative stress and neoplastic transformation in the Bhas 42 cell carcinogenesis assay. All of the polyphenols completely prevented the increased intracellular ROS generation by B[a]P at 12 h, and most inhibited after 3 days. B[a]P increased mitochondrial superoxide generation at 12 h, which was inhibited by the anthocyanins and berberine. B[a]P increased expression of genes related to oxidative stress and inflammation (Nrf2, UCP2, and TNF-α) after 24 h. Polyphenols strongly inhibited the increase in TNF-α and also several polyphenols inhibited the increase in UCP2. At 21 days after 72 h treatment, B[a]P produced a large increase in the number of neoplastic colonies. This transformation was inhibited by most polyphenols, and strongly by resveratrol. In summary, all tested polyphenols were able to inhibit B[a]P-induced increases in markers of oxidative stress and inflammation, and to inhibit cellular transformation, with resveratrol being notable for the strongest preventive effect on cell transformation. The results support a role for dietary polyphenols in protecting against B[a]P-induced carcinogenesis.

Comparison of dietary polyphenols for protection against molecular mechanisms underlying nonalcoholic fatty liver disease in a cell model of steatosis.

SCOPE: Dietary polyphenols have shown promise in protecting the liver against nonalcoholic fatty liver disease. The relative effectiveness and mechanisms of different polyphenols however is mostly unknown. METHODS AND RESULTS: In a model of steatosis using HepG2 hepatocytes, we evaluated the protective effects of different classes of polyphenols and the contributing mechanisms. The treatment of the cells with oleic acid increased reactive oxygen species (ROS) generation and expression of tumor necrosis factor alpha (TNF-α), decreased expression of uncoupling protein 2, and decreased mitochondrial content and markers of biogenesis. The treatment with 1-10 µM polyphenols (resveratrol, quercetin, catechin, cyanidin, kuromanin, and berberine), as well as phenolic degradation products (caffeic acid, protocatechuic acid, and 2,4,6-trihydroxybenzaldehyde), all protected by more than 50% against the oleic acid induced increase in ROS. In other mechanisms involved, the polyphenols except anthocyanins strongly prevented or reversed the effect on mitochondrial content/biogenesis, increased expression of manganese superoxide dismutase, and prevented the large increase in TNF-α expression. Most polyphenols also prevented the decrease in uncoupling protein 2. The anthocyanins were unique in decreasing ROS generation without inducing mitochondrial biogenesis or manganese superoxide dismutase expression. CONCLUSION: While different polyphenols similarly decreased cellular ROS in this model of steatosis, they differed in their ability to suppress TNF-α expression and induce mitochondrial biogenesis and content.

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.

Dietary broccoli sprouts protect against myocardial oxidative damage and cell death during ischemia-reperfusion.

Cruciferous vegetables are known for antioxidant and anti-carcinogenic effects. In the current study we asked whether dietary broccoli sprouts can protect the heart from ischemia-reperfusion. Rats were fed either control diet (sham and control groups) or a diet mixed with 2% dried broccoli sprouts for 10 days. After 10 days the isolated hearts were subjected to ischemia for 20 min and reperfusion for 2 h, and evaluated for cell death, oxidative damage, and Nrf2-regulated phase 2 enzyme activities. Broccoli sprouts feeding inhibited markers of necrosis (lactate dehydrogenase release) and apoptosis (caspase-3 activity) by 78-86%, and decreased indices of oxidative stress (thiobarbituric acid reactive substances and aconitase inactivation) by 82-116%. While broccoli sprouts increased total glutathione and activities of the phase 2 enzymes glutamate cysteine ligase and quinone reductase in liver, they did not affect these in ischemic-reperfused heart. While the mechanism is not clear, the results show that a relatively short dietary treatment with broccoli sprouts can strongly protect the heart against oxidative stress and cell death caused by ischemia-reperfusion.

Dietary green tea extract increases phase 2 enzyme activities in protecting against myocardial ischemia-reperfusion.

Green tea catechins are dietary antioxidant compounds that have been shown to protect against myocardial ischemia-reperfusion (IR) injury. Considering reports that catechins can induce phase 2 enzymes in cultured cells and some organs, we hypothesized that part of the protection to heart against IR injury may involve elevation of phase 2 enzyme activities. Rats were fed for 10 days with either control diet (sham and control groups) or the diet mixed with 0.25% green tea extract. At the end of 10 days, hearts were excised and subjected to global ischemia for 20 min followed by reperfusion for 2 hours. The hearts were compared for indices of cell death, oxidative stress, and phase 2 enzyme activities. Hearts from the green tea group had a 65% to 85% decrease in markers of apoptosis, a tendency to higher total glutathione, and higher activities of the phase 2 enzymes glutamate cysteine ligase and quinone reductase. The results support a possible involvement of phase 2 enzymes in the protection by green tea catechins against myocardial IR injury.

3,5-Bis(benzylidene)-4-oxo-1-phosphonopiperidines and related diethyl esters: Potent cytotoxins with multi-drug-resistance reverting properties.

A series of 3,5-bis(benzylidene)-4-piperidones 3 were converted into the corresponding 3,5-bis(benzylidene)-1-phosphono-4-piperidones 5 via diethyl esters 4. The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T-lymphocytes as well as murine leukemia L1210 cells. In general, the N-phosphono compounds 5, which are more hydrophilic than the analogues in series 3 and 4, were the most potent cluster of cytotoxins, and, in particular, 3,5-bis-(2-nitrobenzylidene)-1-phosphono-4-piperidone 5 g had an average IC(50) value of 34 nM toward the two T-lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC(50) values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi-drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.

The cytotoxic properties and preferential toxicity to tumour cells displayed by some 2,4-bis(benzylidene)-8-methyl-8-azabicyclo[3.2.1] octan-3-ones and 3,5-bis(benzylidene)-1-methyl-4-piperidones.

This study demonstrated that replacement of the axial protons on the C2 and C6 atoms of various 1-methyl-3,5-bis(benzylidene)-4-piperidones 3 by a dimethylene bridge leading to series 2 lowered cytotoxic potencies. Four compounds 2a and 3a-c emerged as lead molecules based on their toxicity towards different neoplasms and their selective toxicity for malignant rather than normal cells. Some possible reasons for the disparity between the IC(50) values in the two series of compounds are presented based on molecular modeling, logP values and respiration in rat liver mitochondria.

E,E-2-Benzylidene-6-(nitrobenzylidene)cyclohexanones: syntheses, cytotoxicity and an examination of some of their electronic, steric, and hydrophobic properties.

Three series of structurally isomeric 2-benzylidene-6-(nitrobenzylidene) cyclohexanones 1-3 were prepared and evaluated against human Molt/C8 and CEM T-lymphocytes as well as murine L1210 cells. The IC(50) values of the majority of compounds are less than 10microM and in some assays, the figures for 1d and 1e are submicromolar. Correlations were discerned between cytotoxic potencies and the atomic charges on one of the olefinic carbon atoms, the torsion angles between an aryl ring, and the adjacent unsaturated group as well as logP values. Three representative compounds were examined for their effect on respiration in rat liver mitochondria.

Oxidative damage to ferritin by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.