Dysobinol from <i>Chisocheton macrophyllus</i> Selectively Induces G1 Cell Cycle Arrest in MCF-7 Breast Cancer Cells

Chrysanthemum boreale is a plant widespread in East Asia, used in folk medicine to treat various disorders, such as pneumonia, colitis, stomatitis, and carbuncle. Whether the essential oil from C. boreale (ECB) and its active constituents have anti-proliferative activities in lung cancer is unknown. Therefore, we investigated the cytotoxic effects of ECB in A549 and NCI-H358 human lung cancer cells. Culture of A549 and NCI-H358 cells with ECB induced apoptotic cell death, as revealed by an increase in annexin V staining. ECB treatment reduced mitochondrial membrane potential (MMP), disrupted the balance between pro-apoptotic and anti-apoptotic Bcl-2 proteins, and activated caspase-8, -9, and -3, as assessed by western blot analysis. Interestingly, pretreatment with a broad-spectrum caspase inhibitor (z-VAD-fmk) significantly attenuated ECB-induced apoptosis. Furthermore, gas chromatography–mass spectrometry (GC/MS) analysis of ECB identified six compounds. Among them, β-caryophyllene exhibited a potent anti-proliferative effect, and thus was identified as the major active compound. β- Caryophyllene induced G1 cell cycle arrest by downregulating cyclin D1, cyclin E, cyclin-dependent protein kinase (CDK) -2, -4, and -6, and RB phosphorylation, and by upregulating p21CIP1/WAF1 and p27KIP1. These results indicate that β-caryophyllene exerts cytotoxic activity in lung cancer cells through induction of cell cycle arrest.

The cell cycle, a vital process that involves in cells' growth and division, lies at the heart of cancer. It has been shown that IB-MECA, an A3 adenosine receptor agonist inhibits the proliferation of cancer cells by inducing cell cycle arrest in several tumors. In this study, we evaluated the role of IB-MECA inhibition in cell cycle progression in ovarian cancer cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in Caov-4 and OVCAR-3. Analysis of cell cycle distribution was carried out by flow cytometry. To determine the mechanisms of IB-MECA-mediated induction of cell cycle arrest, the expression of cell cycle regulatory proteins Cyclin D1 and cyclin-dependent kinase 4 (CDK4) was evaluated. Our results showed that IB-MECA significantly reduced cell viability in a dose-dependent manner. Moreover, our results indicated that a low concentration of IB-MECA induced G1 cell cycle arrest. Reduction of Cyclin D1 and CDK4 protein levels was also observed after treating cancer cells with IB-MECA. This study demonstrated that IB-MECA induces G1 phase cell cycle arrest through Cyclin D1/CDK4-mediated pathway in ovarian cancer cells.

Introduction: Prostate cancer (PCa) often progresses to castration-resistant prostate cancer (CRPC), which is linked to higher treatment resistance and recurrence rates. This highlights the urgent need for new therapeutic options. Natural products, especially flavonoids, have shown promise in reducing drug resistance and possess both antioxidant and anticancer effects. Developing drugs that specifically target CRPC could offer significant therapeutic advantages.Methods: Chrysosplenetin B (CspB) was extracted and purified from the herb Laggera pterodonta (DC.) Benth. using traditional flavonoid extraction techniques, followed by high-performance liquid chromatography (HPLC) for purity assessment and nuclear magnetic resonance (NMR) for structural identification. The effect of CspB on the viability of PCa cells was evaluated using the Cell Counting Kit-8 assay. Subsequently, transcriptome analysis was conducted, and cell cycle progression was assessed through flow cytometry in conjunction with propidium iodide (PI) staining. Additionally, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to confirm the expression levels of relevant proteins and genes.Results: CspB was found to inhibit the proliferation of PC3, DU145, and LNCaP cells in a dose-dependent manner, with a stronger effect noted in PC3 and DU145 cells. Transcriptomic analysis revealed that CspB treatment led to cell cycle arrest, particularly in PC3 cells. Flow cytometry with PI staining confirmed that CspB caused G1 phase cell cycle arrest in PC3 cells. Moreover, CspB treatment significantly increased the expression of essential members of the Cip/Kip family, including CIP1/P21 and KIP1/P27, as well as CDKN2B (P15) and CDKN2D (P19) from the INK4 family. Additionally, CspB exposure notably raised the expression of the G1 phase-negative regulatory gene CDKN1C, while key cell cycle regulators like CDK6 and E2F1 were significantly downregulated at the protein level. Conclusion: Our findings indicate that CspB effectively inhibits the proliferation of CRPC cells by reducing the activity of cell cycle proteins and cyclin-dependent kinase (CDK) complexes while upregulating the expression of P21 and P27 and inducing G1 phase cell cycle arrest. These results highlight the potential of CspB as a promising candidate for developing therapeutic agents aimed at targeting CRPC.

Abstract #5034 Background: Peroxisome proliferator-activated receptor-gamma(PPAR-γ) ligands inhibit cell proliferation and induce apoptosis in cancer cells. And, it is reported that PPAR-γ ligands could serve as negative regulators of breast cancer development and progression, but their mechanism is still unknown. Here we wished to determine whether the PPAR-γ ligand induces cell cycle arrest and apoptosis of MDA-MB-231(ERα-negative) and MCF-7(ERα-positive) breast cancer cell.
 Methods: The effect of PPAR-γ ligands on the cell viability of breast cancer cells was determined using mitochondrial tetrazolium(MTT) assay. The cell cycle distribution and apoptosis induction were evaluated by using the flow cytometry. The expression of apoptosis-related proteins were measured with Western blot analysis. Statistical analysis was performed using Student's t test, and p<0.05 was considered significant.
 Results: The treatment of MDA-MB-231 cell with PPAR-γ ligand, troglitazone was shown to induce cell cycle G1 arrest and induction of apoptosis. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in phosphorylated retinoblastoma(pRb), cyclin D1, D2, D3, cyclin dependent kinase(Cdk) 2, 4, and 6 expression as well as a significant increase in Cdk inhibitor, p21 and p27. Troglitazone showed antiproliferative effect on MCF-7 cell with tamoxifen, respectively and synergically. Troglitazone and tamoxifen could induce G1 arrest and apoptosis of MCF-7 cell, through upregulation of Bax and downregulation of Bcl-2 and cyclin D1.
 Conclusion: PPAR-γ ligand, troglitazone induces cell cycle arrest and apoptosis of MDA-MB-231 cell and increases the sensitivity of anti-hormonal therapy in MCF-7 cell. These results suggest that troglitazone has anticancer effect on both ERα-negative and positive breast cancer cells. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5034.

Glioblastoma multiforme (GBM) is among the most aggressive and challenging brain tumors, with limited treatment options. Cimicifuga foetida, a traditional Chinese medicine, has shown promise due to its bioactive components. This study investigates the anti-glioma effects of a methanolic extract of C. foetida (CF-ME) in GBM cell lines. The effects of CF-ME and its index compounds (caffeic acid, cimifugin, ferulic acid, and isoferulic acid) on GBM cell viability were assessed using MTT assays on U87 MG, A172, and T98G cell lines. The ability of CF-ME to induce cell cycle arrest, apoptosis, and autophagy and inhibit metastasis was evaluated using flow cytometry, Western blotting, and functional assays. Additionally, the synergistic potential of CF-ME with temozolomide (TMZ) was explored. CF-ME significantly reduced GBM cell viability in a dose- and time-dependent manner, induced G1 phase cell cycle arrest, promoted apoptosis via caspase activation, and triggered autophagy. CF-ME also inhibited GBM cell invasion, migration, and adhesion, likely by modulating epithelial-mesenchymal transition (EMT) markers. Combined with TMZ, CF-ME further enhanced reduced GBM cell viability, suggesting a potential synergistic effect. However, the individual index compounds of CF-ME exhibited only modest inhibitory effects, indicating that the full anti-glioma activity may result from the synergistic interactions among its components. CF-ME exhibited potent anti-glioma activity through multiple mechanisms, including cell cycle arrest, apoptosis, autophagy, and the inhibition of metastasis. Combining CF-ME with TMZ further enhanced its therapeutic potential, making it a promising candidate for adjuvant therapy in glioblastoma treatment.

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit the enzyme cyclooxygenase (COX), are known to have a potent anti-tumorigenic activity in various cancers. However, the responsible molecular mechanisms of COX inhibition in breast cancer cells remain to be completely elucidated. We examined the effect of the selective COX-1 inhibitor, FR122047 and the selective COX-2 inhibitor, SC791 on cell growth and apoptosis in human breast cancer MCF-7 cells which exhibited a high basal level of COX-1 expression. Compared to SC791, FR122047 treatment led to a distinct suppression of cell growth in MCF-7 cells. Upon FR122047 treatment, there were apparent increases in the ratio of Bax to Bcl-2, mitochondrial cytochrome c release, and apoptosis in MCF-7 cells. Our data showed that treatment of caspase-8 inhibitor could significantly suppress the cleavage of the effector caspase-7 and PARP in FR122047-treated MCF-7 cells which are caspase-3-deficient breast cancer cells, indicating that the induction of apoptosis by FR122047 is significantly dependent on caspase-8 activity in MCF-7 breast cancer cells. Our data suggest that the NSAID FR122047 may have an anti-cancer potential in breast cancer.

Nannocystin ax, an eEF1A inhibitor, induces G1 cell cycle arrest and caspase-independent apoptosis through cyclin D1 downregulation in colon cancer in vivo

BackgroundDillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells.MethodsDillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes.ResultsDCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 μg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 μg/mL) and high concentration (50 μg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells.ConclusionDCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.

Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the dual-specificity protein phosphatase family, which is hypothesized to regulate cell cycle progression in tumor cells. However, whether CDKN3 is a potential therapeutic target for breast cancer remains to be elucidated. The present in vitro study aimed to investigate the potential roles of CDKN3 in breast cancer. Breast cancer cell lines were used to detect CDKN3 expression, and CDKN3 expression was silenced to investigate its role in cell apoptosis, cell cycle arrest and migration. The underlying mechanisms were screened by detecting proliferating cell nuclear antigen (PCNA), Ras homolog gene family, member A (RhoA), vimentin, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) expression. CDKN3 was highly expressed in MCF-7 and BT474 cell lines. The silencing of CDKN3 in MCF-7 and BT474 cell lines promoted cell apoptosis, induced G1 phase cell cycle arrest and inhibited cell migration. The expression levels of PCNA, RhoA, vimentin and Bcl-2 were downregulated following CDKN3 silencing. Conversely, Bax expression was increased, as compared with the vehicle control. These results suggest that CDKN3 acts as an oncogene during breast cancer progression. The in vitro silencing of CDKN3 promoted apoptosis, induced G1 phase cell cycle arrest and inhibited cell migration. Possible mechanisms are associated with the regulation of PCNA, Bcl-2, vimentin, RhoA and Bax expression. CDKN3 may therefore be considered a potential target for the treatment of breast cancer.

Breast cancer is most frequently detected and leading cause of cancer death in women worldwide. 80% of the breast cancer are estrogen receptor positive and the presently available drugs are ineffective either due to intrinsic resistance or due to acquired resistance. Sulfonamide compounds are a class of compounds showing activities like, antibacterial, antiviral including antitumor. In the present study we have investigated the anti-cancerous activity of sulfonamide derivative CID-6861424 on breast cancer cell line, MCF-7 . Our data shows inhibition of MCF-7 cell viability by CID-6861424 in a concentration and time dependent manner. 50μM was the IC 50 value of CID-6861424 on MCF-7 cell. The compound downregulated cyclin D1 and CDK 4/6, induced G 1 phase cell cycle arrest and apoptotic cell death. 50μM CID-6861424 upregulated ROS generation, disrupted mitochondrial membrane potential (Δ ψm ), increased DNA damage and upregulated tumor suppressor protein, p53 in MCF-7 cells. The compound reduced phosphorylation of Akt and GSK-3β, downregulated Bcl-2 and upregulated Bax indicating apoptotic cell death. These results suggest CID-6861424 as a potential anticancer agent against breast cancer. Keywords: CID-6861424 ; MCF-7 cell, cell cycle, double-strand DNA break, apoptosis. Cite this Article Sumit Kumar Gautam, Afreen Inam, Amir Azam, Neelima Mondal. Induction of G1 phase cell cycle arrest and apoptosis in breast cancer MCF-7 cells by sulphonamide derivative CID-6861424. Research & Reviews: A Journal of Toxicology . 2019; 9(2): 1–19p.

Hypoxia is a well-recognized clinical feature of solid tumors, including lung cancer, and is associated with poor prognosis due to its role in promoting resistance to chemotherapy and radiotherapy. To investigate the cellular consequences of hypoxia, we cultured A549 lung adenocarcinoma cells under 1% O2 and examined their growth, cell cycle distribution, and redox status. Hypoxia significantly reduces cell proliferation and induced G1 phase cell cycle arrest, suggesting a cytostatic effect. Biochemical analysis showed a 2.64- and 2.04-fold increase in total and mitochondrial reactive oxygen species (ROS) levels, respectively, along with an elevated total thiol levels under hypoxic conditions compared to normoxia. To assess the reversibility of the hypoxic response, we performed a reciprocal oxygen exposure experiment where cells initially grown under hypoxia were re-exposed to normoxia, and vice versa. To explore the underlying molecular mechanism, we analyzed transcriptomic datasets (GEO accession: GSE48134 and GSE42416) which revealed that hypoxia downregulated key genes involved in energy metabolism (e.g., PDK4, G6PD), cell cycle progression (e.g., CCND1, CDK2), and redox regulation (e.g., GCLM, TXNRD1, NQO1, GCLC). Further, few of redox-related genes were validated by RT-PCR in A549 cells cultured under hypoxia and normoxia for 24 h. Importantly, cyclic hypoxia (intermittent hypoxia-reoxygenation) conditions showed partial restoration of some of these transcripts, supporting the transient nature of hypoxic stress, consistent with our in vitro observations. Furthermore, transcriptome profiles from adenocarcinoma patients (GEO accession: GSE30979) also match our cell line observations. Thus, our results clearly show that hypoxia causes a temporary cell cycle arrest in lung cancer cells, which is reversible when oxygen is restored.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12032-025-03058-9.

Pinus densiflora sieb. et zucc.(pine needle) is a traditional medicine used in several East Asian countries. However, the efficacy of pine needle has rarely been reported. In this study showed that the anti-proliferative effects and the mechanisms of hexane layer of pine needle MeOH extract (PNH) on gastric cancer cells. At first, PNH inhibited the proliferation of gastric cancer cells in a dose-dependent manner. Moreover, PNH treatment induced G1 phase cell cycle arrest through the increased p27KIP1 expression and decreased cyclin dependent kinase (CDKs) activity. Furthermore, PNH treatment induced premature senescence without oncogenic stress, through the expression of p27KIP1 and Skp2. Taken together, these results showed that PNH inhibited gastric cancer cell proliferation through the induction of G1-cell cycle arrest and premature senescence via induced p27KIP1 expression, as controlled by Skp2 reduction. Also, PNH could be a candidate for anti-gastric cancer treatment and may be useful in the development of anti-gastric cancer drugs.

1. This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. 2. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF-7, MCF-7ras, T47-D, and BT-20 cells, as well as arrest in G2/M in MDA-MB-231 cells. 3. Transient transfection of MCF-7 and T47-D cells with wild-type and antisense p53 did not modify butyrate-induced apoptosis. Pifithrin-alpha, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF-7 and T47-D cells treated with butyrate. These results indicate that P53 was not involved in butyrate-induced growth inhibition of breast cancer cells. 4. Treatment of MCF-7 cells with anti-Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas-induced apoptosis, as massive apoptosis was observed rapidly when MCF-7 cells were treated with butyrate and anti-Fas agonist antibody. In addition, butyrate-induced apoptosis in MCF-7 cells was considerably reduced by anti-Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate-induced apoptosis may be mediated by Fas signalling. 5. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53-independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas-triggered apoptosis in MCF-7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy.

Copper complexes have the potential to be developed as targeted therapy for cancer because cancer cells take up larger amounts of copper than normal cells. Copper complex Cu(SBCM)2 has been reported to induce cell cycle arrest and apoptosis towards triple-negative breast cancer cells. Nevertheless, its effect towards other breast cancer subtypes has not been explored. Therefore, the present study was conducted to investigate the effect of Cu(SBCM)2 towards oestrogen-receptor positive MCF-7 breast cancer cells. Growth inhibition of Cu(SBCM)2 towards MCF-7 and human non-cancerous MCF-10A breast cells was determined by MTT assay. Morphological changes of Cu(SBCM)2-treated-MCF-7 cells were observed under an inverted microscope. Annexin V/PI apoptosis assay and cell cycle analysis were evaluated by flow cytometry. The expression of wild-type p53 protein was evaluated by Western blot analysis. The intracellular ROS levels of MCF-7 treated with Cu(SBCM)2 were detected using DCFH-DA under a fluorescence microscope. The cells were then co-treated with Cu(SBCM)2 and antioxidants to evaluate the involvement of ROS in the cytotoxicity of Cu(SBCM)2. Docking studies of Cu(SBCM)2 with DNA, DNA topoisomerase I, and human ribonucleotide reductase were also performed. The growth of MCF-7 cells was inhibited by Cu(SBCM)2 in a dose-dependent manner with less toxicity towards MCF-10A cells. It was found that Cu(SBCM)2 induced G2/M cell cycle arrest and apoptosis in MCF-7 cells, possibly via a p53 pathway. Induction of intracellular ROS was not detected in MCF-7 cells. Interestingly, antioxidants enhance the cytotoxicity of Cu(SBCM)2 towards MCF-7 cells. DNA topoisomerase I may be the most likely target that accounts for the cytotoxicity of Cu(SBCM)2.

The synthesis of a series of novel N-substituted tetrahydro-β-carboline-imidazolium salt derivatives is presented. The biological properties of the compounds were evaluated in vitro against a panel of human tumor cell lines. The results suggest that the benzimidazole ring and 1-(naphthalen-2-yl)ethan-1-one or 2-naphthylmethyl substituent at the imidazolyl-3-position were vital for modulating cytotoxic activity. Compound 41 was observed as a potent derivative with IC50 values of 3.24-8.78 μM and exhibited cytotoxic activity selectively against HL-60, A-549 and MCF-7 cell lines. Meanwhile, high inhibitory activities selectively against HL-60 and MCF-7 cell lines were observed for compound 51. Moreover, compound 51 was able to induce G1 phase cell cycle arrest and apoptosis in MCF-7 cells. The cytotoxicity of compound 51 against human normal lung epithelial cell line BEAS-2B was further evaluated.