Acid Induces Cell Cycle Arrest Research Articles

Abstract 4722: Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System

Abstract Phenotypic screening is a valuable tool for analysis of the complex mix of effects that drug treatment has on cells. Traditionally image-based screening uses fixed cells stained with reagents at a pre-chosen end point, however our technique combines kinetic, label-free readouts with non-perturbing fluorescence reporters to enable information-rich quantification of compound effects. A549 lung cancer cells were treated with a library of over 800 FDA-approved drugs while images were acquired in the Incucyte® Live-Cell Analysis System every two hours for four days. Cell growth and viability were measured using label-free readouts of confluence and dead cell count while cell cycle, cell death and Akt pathways were investigated using multiplexed fluorescent readouts. Cytotoxicity was measured using label-free Incucyte® AI Cell Health Analysis Software Module which uses two neural networks to segment individual cells and classify them as live or dead. Assay robustness was quantified using the time course of positive and negative controls (camptothecin and vehicle, respectively). Z’ reached a maximal value >0.9 between 48h and 72h; at 72h 4.4% of the compounds within the library induced >50% cell death. Correlation between % Live cells and % Confluence indicated 3 types of effect: cytotoxic (low viability, low confluence), cytostatic (high viability, low confluence), and none (high viability, high confluence). Morphological changes were observed in a multitude of wells with 29 compounds inducing cell enlargement >1800 µm2, suggesting induction of senescence. Hits from fluorescent readouts were identified as the compounds which perturbed the quantified readout more than 3 standard deviations away from the mean value of the vehicle. Mechanisms of cell death were examined by correlative analysis of the total dead cell population (label-free cell death identification), caspase-active (caspase 3/7-reagent positive) and Annexin V positive cells. This enabled rapid identification of compounds which induced apoptosis by caspase-independent pathways, including the serotonin-selective reuptake inhibitor Sertraline. Kinetic plots of cell cycle stage (% S|G2|M or G1) revealed contrasting mechanisms. Mycophenolic acid induced cell cycle arrest, observed as a consistent increase in the percentage of cells in G1 over time. However flumazenil displayed repeated, transient peaks in the percentage of cells in S|G2|M over the same 72h period, indicating synchronisation behavior. Incucyte® Live-Cell Analysis System as a phenotypic screening platform provides valuable kinetic data from cells within the physiologically relevant environment of a standard incubator. Label-free readouts yield direct cellular information and can be combined with fluorescent data to provide vastly more information than a single endpoint readout. Citation Format: Kirsty McBain, Gillian Lovell, Jasmine Trigg, Nicola Bevan, Daniel Appledorn. Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4722.

P13-048-23 Ursodeoxycholic Acid Induces Cell Cycle Arrest and Apoptosis in Cancerous HCT116 Colon Cells but to a Lesser Extent in Noncancerous NCM460 Colon Cells

P13-048-23 Ursodeoxycholic Acid Induces Cell Cycle Arrest and Apoptosis in Cancerous HCT116 Colon Cells but to a Lesser Extent in Noncancerous NCM460 Colon Cells

Apoptotic and cytostatic actions of maslinic acid in colorectal cancer cells through possible IKK-β inhibition

Objective: To explore the anti-cancer activity of maslinic acid against colorectal cancer (CRC) cell lines and its possible mechanism. Methods: The inhibitory effect of maslinic acid was screened against five CRC cell lines (HT-29, HCT 116, SW480, SW48, and LS 174T) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle analyses were carried out using annexin V-FITC/propidium iodide staining and propidium iodide staining, respectively and subjected to fluorescence-activated cell sorting analysis. Protein expression studies of inhibitor of κB kinase-β (IKK-β), checkpoint kinase 1 (Chk1) and cyclin D1 were conducted using the JESS system. Results: Maslinic acid exhibited growth inhibitory effect in a dose- and time-dependent manner in HT-29 and HCT 116 cell lines. A more prominent apoptosis induced by maslinic acid was observed in HCT 116 cell line. However, in HT-29 cell line, maslinic acid induced cell cycle arrest by inhibiting the G1S transition, which was accompanied by the downregulation of cyclin D1. The expression of unphosphorylated IKK-β protein was increased in both (HT-29 and HCT 116) cell lines after maslinic acid treatment. Conclusions: Maslinic acid inhibits the growth of HT-29 and HCT 116 cells in a different manner, induces cell cycle arrest in HT-29 cells and causes apoptosis in HCT 116 cells partially via NF-κB pathway inhibition.

Abstract PO-132: Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells

Abstract Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including anti-inflammatory, immunomodulatory, anti-mutagenic, antioxidant, and anticancer activities. This study evaluated the effects of RA in two racially different triple-negative breast cancer (TNBC) cell lines. One cell line was derived from Caucasian origin (MDA-MB-231), and another one was derived from an African American origin (MDA-MB-468). The apoptotic effect of RA was performed using An Annexin V-FITC Apoptosis Detection Kit, while the Propidium Iodide Flow Cytometry Kit was used to detect the cell cycle distribution. Gene expression profiling was established using Human Apoptosis PCR array. The results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. It was noted that RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Furthermore, in MDA-MB- 231 cells, RA significantly upregulated the mRNA expression of three genes: Harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, RA induced a significant transcription activation in three genes, including TNF, growth arrest, and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in African American cells. Citation Format: Karam F.A. Soliman, Samia S. Messeha, Najla O. Zarmouh, Abrar Asiri. Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-132.

18β-glycyrrhetinic Acid Induces Cell Cycle Arrest and Apoptosis in HPV18+ HeLa Cervical Cancer Cells

Aims: In recent years, natural products have received great attention to cancer prevention owing to their various health benefits, lack of toxicity, and side effects. Accumulating evidence shows that 18β-glycyrrhetinic acid (GRA) has antiproliferative and apoptotic activities on many cancer cell lines, while its role in cervical cancer remains unknown. Thus, the current research was conducted to illustrate GRA's cytotoxic effect against the HeLa cell line of HPV18 + human cervical cancer.
 Methodology: The effect of GRA on HeLa cell line was tested by MTT and Trypan blue dye exclusion assay. Cell cycle analysis was carried out by flow cytometry after PI staining. Apoptosis was assessed after annexin V / PI double staining by flow cytometry. The Caspase activation assay kit analysed caspase activation. Reactive oxygen species (ROS) generation was measured by fluorimeter after DCFDA dye staining.
 Results: Results of the current study have shown that GRA exposure significantly inhibited the cell viability of HeLa cells in a dose- and time-dependent manner. GRA induced growth arrest of HeLa cells at G0/G1 phase of the cell cycle. Moreover, GRA's antiproliferative action was mediated through apoptosis, as evident from caspase-3 and -9 activation. Caspase inhibitors blocked the GRA-induced caspase activation and ameliorated the GRA-induced cytotoxicity. This suggested the role of the intrinsic pathway of apoptosis stimulated by GRA. The intracellular ROS generation assay showed a dose-related increment in ROS production induced by GRA. Co-culturing of HeLa cells with N-acetyl cysteine (NAC), a ROS inhibitor, completely abrogated GRA-induced cell cycle arrest and apoptosis. Thus, the effect of NAC suggested the involvement of intracellular ROS in the GRA-induced cytotoxicity.
 Conclusion: In summary, GRA exhibited strong antiproliferative and apoptotic properties and, thus, could act as an adjunct in the prevention and management of cervical cancer.

Kaurenoic Acid Induces Cell Cycle Arrest and Apoptosis in the MCF‐7 Breast Cancer Cell Line

AbstractBreast cancer continues to be the major cause of cancer death among women. The high genetic heterogeneity and chemoresistant phenotypes of breast tumors require the search for new compounds to expand the options for the treatment of this disease. Kaurenoic acid (KA) is an ent‐kaurane type‐diterpene with cytotoxic and genotoxic effects against gastric and cervical cancer cells. In the present study, we report the antiproliferative effects of KA in the breast tumoral cell line MCF‐7 and the normal breast counterpart MCF‐10 A. The long‐term cytotoxic effects of KA were observed by the reduction of survival fraction at 50 and 100 μM. At these concentrations, the treatment with KA increased the extension of DNA damage followed by G1 cell cycle phase accumulation and induction of apoptosis in both cell lines. Taken together, the results contained herein support the promising antiproliferative effects of KA.

Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including, anti-inflammatory, immunomodulatory, and neuroprotective, as well as having antioxidant and anticancer activities. This study evaluated the effects and mechanisms of RA in two racially different triple-negative breast cancer (TNBC) cell lines. Results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. Remarkably, RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Further, in MDA-MB-231 cells, RA significantly upregulated the mRNA expression of three genes: harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, the compound induced a significant transcription activation in three genes, including TNF, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in MDA-MB-468 cells.

HDAC Inhibition Induces PD-L1 Expression in a Novel Anaplastic Thyroid Cancer Cell Line

While papillary thyroid cancer (PTC) has largely favorable prognosis, anaplastic thyroid cancer (ATC) is a rare but extremely aggressive malignancy with grim clinical outcome. Even though new therapeutic options are emerging for ATC, additional preclinical models and novel combinations are needed for specific subsets of patients. We established a novel cell line (PF49) from the malignant pleural effusion of a 68-year-old male patient with ATC that rapidly transformed from a BRAF and TERT promoter mutant PTC. PF49 cells demonstrated a robust migratory activity in vitro and strong invasive capacity in vivo in a pleural carcinosis model. Combined BRAF and MEK inhibition decreased the proliferation and migration of PF49 cells, however could not induce cell death. Importantly, HDAC inhibitor treatment with SAHA or valproic acid induced cell cycle arrest and strongly increased PD-L1 expression of the tumor cells. Induction of PD-L1 expression was also present when paclitaxel-cisplatin chemotherapeutic treatment was combined with HDAC inhibitor treatment. Increased PD-L1 expression after HDAC inhibition was recapitulated in an international ATC cell model. Our data suggest that HDAC inhibition alone or in combination with standard chemotherapy may potentiate anaplastic thyroid cancer cells for immunotherapy.

Ellagic acid induces cell cycle arrest and apoptosis via the TGF‑β1/Smad3 signaling pathway in human colon cancer HCT‑116 cells.

Colorectal carcinoma (CRC) is a major type of malignancy worldwide. Ellagic acid (EA), a natural phenolic constituent, has been shown to exhibit anticancer effects. In our previous study, it was shown that EA inhibited proliferation of CRC cells. Additionally, microarray analysis revealed 4,738 differentially expressed genes (DEGs) which were associated with multiple cellular events, including cell growth, apoptosis and angiogenesis. However, the associated pathways had not been validated. In the present study, it was shown that EA induced G0/G1 cell cycle arrest in HCT‑116 cells, and increased apoptosis. Furthermore, DEGs identified by cDNA microarray analysis were investigated, and showed changes in five genes which were associated with the TGF‑β1/Smad3 signaling pathway. TGF‑β1 small interfering RNA and SIS3, a Smad3 inhibitor, were used to assess the role of TGF‑β1 and Smad3, respectively, and it was shown that the they reduced the effects of EA on HCT‑116 CRC cells. In addition, the expression patterns of downstream DEGs of the TGF‑β1/Smad3 pathway were altered. Thus, this pathway may underlie the molecular mechanism by which EA exhibits its effects invitro in CRC cells. Accordingly, targeting the TGF‑β1/Smad3 pathway with anticancer agents such as EA may be potentially used to treat CRC.

Corosolic acid induces cell cycle arrest and cell apoptosis in human retinoblastoma Y-79 cells via disruption of MELK-FoxM1 signaling.

Retinoblastoma(Rb) is the most frequent primary intraocular tumor usually diagnosed in infants and children, and current therapy for such disease is still limited. Corosolic acid(CA), an ursane-type pentacyclic triterpene, has been assessed as a promising anticancer agent with little impact on untransformed cells. In the present study, we investigated the cytotoxic effect and underlying mechanism of CA on human retinoblastoma Y-79 cells. The viability of cells was verified by MTT assay. Cell cycle and apoptosis were evaluated by flow cytometric analysis. The expressions and activities of the related molecules were assessed by western blot analysis and luciferase assay. The results demonstrated that the treatment of CA dose-dependently induced cytotoxicity, cell cycle arrest and cell apoptosis in Y-79 cells. Furthermore, MELK-FoxM1 signaling was estimated to be involved in the cytotoxic effect of CA on Y-79 cells, and CA exerted its activity mainly through inhibition of the expression levels of MELK and FoxM1 as well as through suppression of the transcriptional activity of FoxM1 driven by itself or MELK. Our findings establish MELK-FoxM1 signaling as a promising therapeutic target for human retinoblastoma, and suggest the potential development of CA and its derivatives as novel drug candidates against this disease.

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in Candida albicans

BackgroundChlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in Candida albicans. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels. MethodsTo evaluate the inhibitory effects on CRA-induced cell death, C. albicans cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL. ResultsCRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in C. albicans. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked. ConclusionsCRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in C. albicans. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes. General significanceThis study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis.

Abstract 2739: Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo

Abstract Carnosic acid (CA), one of the purified components of rosemary, has shown anti-inflammatory, chemo-preventive, and anticancer activities. In this study, we treated melanoma cell line B16F10 with CA and detected the anticancer effects. After 24-hour treatment, CA decreased cell proliferation rate, wound healing activity, and inhibited colony formation of B16F10. Additionally, CA induced G0/G1 cell cycle arrest, up-regulated p21 expression, and down-regulate cyclin E, cyclin D and CDK4 expression. Carmustine (BCNU) and Lomustine (CCNU) are used in clinical treatment of melanoma. To assay the combinational effects of CA and BCNU or CCNU, B16F10 cells were treated with CA, BCNU, CCNU alone, or CA combined with BCNU or CCNU. Our results showed, BCNU and CCNU arrested cell cycle at G2/M phase after 24-hour treatment. Dramatically, CA enhenced BCNU- and CCNU-induced cell cycle arrest at G2/M phase, and decreased cyclin B1 and p-CDK1 expresion. In the in vivo xenograft model, B16F10 cells (5×105) were inoculated subcutaneously in C57BL/6 mice, and the tumor grew for one week. The mice were divided into six groups and provided different treated strategies: control, CA (50 mg/kg) alone, BCNU (50 mg/kg) alone, CCNU (25 mg/kg) alone, CA combined with BCNU, and CA combined with CCNU. After two-week treatment, compared with control group, CA had better treated effects than BCNU or CCNU. Compared with CA, BCNU or CCNU treatment alone, the combined strategies inhibited the tumor growth in melanoma mice model more effectively. Furthermore, BCNU and CCNU treatment in melanoma mice caused high values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). However, CA significantly decreased AST and ALT values caused by BCNU and CCNU in combined trestment group. Therefore, CA is more safe and effective than BCNU and CCNU, and the combined treatment strategies of CA and BCNU or CCNU result in an increased antitumor efficacy. CA may be suitable for translation to future clinic. Citation Format: Kun-I Lin, Chih-Chien Lin, Ming-Feng Chen, Chang-Han Chen, Li-Yen Shiu. Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2739.

Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity.

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL-sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (qmAb ) in a dose-dependent manner. The beneficial effect of valeric acid on culture longevity and qmAb outweighed its detrimental effect on μ, resulting in 2.9-fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.

Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine-123 DNA-binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose-dependent, as well as time-dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub-G1 (apoptotic) phase of the cell cycle, in a dose-dependent manner. Staining with Annexin V-fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose-dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose-dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC.

Ursolic acid induces cell cycle arrest and apoptosis of gallbladder carcinoma cells.

BackgroundUrsolic acid (UA), a plant extract used in traditional Chinese medicine, exhibits potential anticancer effects in various human cancer cell lines in vitro. In the present study, we evaluated the anti-tumoral properties of UA against gallbladder carcinoma and investigated the potential mechanisms responsible for its effects on proliferation, cell cycle arrest and apoptosis in vitro.MethodsThe anti-tumor activity of UA against GBC-SD and SGC-996 cells was assessed using MTT and colony formation assays. An annexin V/PI double-staining assay was used to detect cell apoptosis. Cell cycle changes were detected using flow cytometry. Rhodamine 123 staining was used to assess the mitochondrial membrane potential (ΔΨm) and validate UA’s ability to induce apoptosis in both cell lines. The effectiveness of UA in gallbladder cancer was further verified in vivo by establishing a xenograft GBC model in nude mice. Finally, the expression levels of cell cycle- and apoptosis-related proteins were analyzed by western blotting.ResultsOur results suggest that UA can significantly inhibit the growth of gallbladder cancer cells. MTT and colony formation assays indicated dose-dependent decreases in cell proliferation. S-phase arrest was observed in both cell lines after treatment with UA. Annexin V/PI staining suggested that UA induced both early and late phases of apoptosis. UA also decreased ΔΨm and altered the expression of molecules regulating the cell cycle and apoptosis. In vivo study showed intraperitoneally injection of UA can significantly inhibited the growth of xenograft tumor in nude mice and the inhibition efficiency is dose related. Activation of caspase-3,-9 and PARP indicated that mitochondrial pathways may be involved in UA-induced apoptosis.ConclusionsTaken together, these results suggest that UA exhibits significant anti-tumor effects by suppressing cell proliferation, promoting apoptosis and inducing 7cell cycle arrest both in vitro and in vivo. It may be a potential agent for treating gallbladder cancer.

Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin.

Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

Superior activity of a new histone deacetylase inhibitor (ZYJ-34c) in inhibiting growth of human leukemia cells by inducing p21 WAF1 expression and cell cycle arrest

Histone deacetylase inhibitors are a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis and cell cycle arrest in various cancer cells. Recently, we identified ZYJ-34c, a modified histone deacetylase inhibitor that showed significantly higher antitumor activity in vivo than the FDA-approved drug suberoylanilide hydroxamic acid. We aimed to investigate its exact mechanism of action. Activity of ZY-34c against human erythroleukemic (K562) cells, human myeloid leukemia (HL-60) cells, and primary leukemic cells were investigated in vitro using proliferation assays, cell cycle assays, apoptosis assays, RNA interference, promoter acetylation assays, and assays of transcription of related molecules. ZYJ-34c strongly inhibited the proliferation of leukemia cells compared with suberoylanilide hydroxamic acid. Primary leukemic cells isolated from patients with acute myeloid leukemia were more sensitive to ZYJ-34c. The 50% growth-inhibitory concentrations of ZYJ-34c and suberoylanilide hydroxamic acid were 2.95±0.75 and 4.45±0.29 μmol/l for K562 cells treated for 48 h, respectively. We found that ZYJ-34c caused more significant G1 cell cycle arrest than suberoylanilide hydroxamic acid, in a time-dependent manner. ZYJ-34c-induced hyperacetylation of histone H3 and H4 around the promoter region of p21. P21 RNA interference markedly impaired ZYJ-34c-induced or suberoylanilide hydroxamic acid-induced G1 cell cycle arrest. In addition, levels of bcr-abl mRNA and protein in K562 cells decreased after treatment with either suberoylanilide hydroxamic acid or ZYJ-34c; moreover, ZYJ-34c had a higher inhibition activity. ZYJ-34c could represent a novel pharmacological agent with potential benefit for patients with leukemia.

Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

MiR-192 Induces G2/M Growth Arrest in Aristolochic Acid Nephropathy

Aristolochic acid nephropathy is characterized by rapidly progressive tubulointerstitial nephritis culminating in end-stage renal failure and urothelial malignancy. Profibrotic effects of aristolochic acid are linked to growth arrest of proximal tubular epithelial cells; however, the underlying mechanisms are largely undetermined. miRNAs are small, endogenous, post-transcriptional regulators of gene expression implicated in numerous physiological and pathological processes. In the present study, we characterized the mechanism of aristolochic acid-induced cell cycle arrest and its regulation by miRNAs. Incubation with aristolochic acid led to profound G2/M arrest in proximal tubular epithelial cells via p53-mediated inactivation of the maturation-promoting complex, CDK1/cyclin-B1. Analysis of miRNA expression identified up-regulation of miRNAs, including miR-192, miR-194, miR-450a, and miR-542-3p. The stable overexpression of miR-192 recapitulated G2/M arrest via repression of the E3 ubiquitin ligase, murine double-minute 2, a negative regulator of p53. p53-induced transcription of p21(cip1) and growth arrest and DNA damage 45 and resulted in the inactivation and dissociation of the maturation-promoting complex. These data demonstrate a core role for miR-192 in mediating proximal tubular epithelial cell G2/M arrest after toxic injury by aristolochic acid. Because numerous studies have linked such growth arrest to fibrosis after proximal tubular epithelial cell injury, this mechanism may have widespread relevance to recovery/nonrecovery after acute kidney injury.

In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid

Ethnopharmacological relevanceWrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far. Aim of the studyTo evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer. Material and methodsPowdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231. ResultsThe ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC50 of 50μg/ml and 30μg/ml for 24h, 28μg/ml and 22μg/ml for 48h and 25μg/ml and 20μg/ml for 72h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC50 value of 7.5μM and 7.0μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts. ConclusionOur results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.