Mechanism Of Cell Cycle Arrest Research Articles

Synthesis, characterization, cellular uptake and apoptosis-inducing properties of two highly cytotoxic cyclometalated ruthenium(II) β-carboline complexes

Two new cyclometalated Ru(II) complexes of the general formula [Ru(N-N)2(1-Ph-βC)](PF6), where N–N = 4,4′-dimethyl-2,2′-bipyridine (dmb, Ru1), 2,2′-bipyridine (bpy, Ru2), and 1-Ph-βC (1-phenyl-9H-pyrido[3,4-b]indole) is a β-carboline alkaloids derivatives, have been synthesized and characterized. The in vitro cytotoxicities, cellular uptake and localization, cell cycle arrest and apoptosis-inducing mechanisms of these complexes have been extensively explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, inductively coupled plasma mass spectrometry (ICP-MS), flow cytometry, comet assay, inverted fluorescence microscope as well as western blotting experimental techniques. Notably, Ru1 and Ru2 exhibit potent antiproliferative activities against selected human cancer cell lines with IC50 values lower than those of cisplatin and other non-cyclometalated Ru(II) β-carboline complexes. The cellular uptake and localization exhibit that these complexes can accumulate in the cell nuclei. Further antitumor mechanism studies show that Ru1 and Ru2 can cause cell cycle arrest in the G0/G1 phase by regulating cell cycle relative proteins and induce apoptosis through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation and ROS-mediated DNA damage.

Molecular mechanism of G2 phase cell cycle arrest in tumor cells

The formation and progression of tumor is inseparable from the changes of cell cycle regulatory proteins. The G2-M period block effect of tumor cells is especially important in slowing the growth of tumor and improving the curative effect. DNA damage receptor, signal transduction factor and effectors are the classical pathways to mediate G2 phase arrest of tumor cells, and have the potential to be new targets for tumor therapy. Key words: DNA damage; Cell cycle; Neoplasms

Novel tetrahydroacridine and cyclopentaquinoline derivatives with fluorobenzoic acid moiety induce cell cycle arrest and apoptosis in lung cancer cells by activation of DNA damage signaling

Lung cancer is still the leading cause of cancer-related death worldwide, indicating a necessity to develop more effective therapy. Acridine derivatives are potential anticancer agents due to their ability to intercalate DNA as well as inhibit enzymes involved in replication and transcription. Recently, we have evaluated anticancer activity of 32 novel acridine-based compounds. We found that the most effective were tetrahydroacridine and cyclopentaquinoline derivatives with fluorobenzoic acid containing eight and nine carbon atoms in the aliphatic chain. The aim of this study was to determine the molecular mechanisms of compounds-induced cell cycle arrest and apoptosis in human lung adenocarcinoma cells. All compounds activated Ataxia telangiectasia mutated kinase and phosphorylated histone H2A.X at Ser139 indicating DNA damage. Treatment of cells with the compounds increased phosphorylation and accumulation of p53 that regulate cell cycle as well as apoptosis. All compounds induced G0/1 cell cycle arrest by phosphorylation of cyclin-dependent kinase 2 at Tyr15 resulting in attenuation of the kinase activity. In addition, cyclopentaquinoline derivatives induced expression of cyclin-dependent kinase 2 inhibitor, p21; however, tetrahydroacridine derivatives had no significant effect on p21. Moreover, all compounds decreased the mitochondrial membrane potential accompanied by increased expression of Bax and down-regulation of Bcl-2, suggesting activation of the mitochondrial pathway. All compounds also significantly attenuated the migration rates of lung cancer cells. Collectively, our findings suggest a central role of activation of DNA damage signaling in response to new acridine derivatives treatment to induce cell cycle arrest and apoptosis in cancer cells and provide support for their further development as potential drug candidates.

Ellagic acid and Annona muricata in the chemoprevention of HPV-related pre-neoplastic lesions of the cervix.

Ellagic acid is a phenolic compound naturally present in nuts and berries. Several studies have demonstrated that this bioactive compound has antioxidant, chemopreventive and antiviral activity. Annona muricata is a type of fruit tree with a long history of traditional use. A number of properties have been attributed to different parts of the plant, including anticancer and antioxidant activities. In the current study, a complex based on ellagic acid, Annona Muricata and antioxidant factors (an ellagic acid complex) was administered to a group of human papilloma virus (HPV) infected women with and without cervical lesions, for 12 months. Its effect on HPV clearance and cervical cytological outcomes was assessed and a group of women with the same clinical features who did not receive the ellagic acid complex served as a control. A positive correlation was observed between intake of ellagic acid complex and negative Pap test following 6 and 12 months of treatment (χ2 test: 0.041 and 0.014, respectively). Women treated with the ellagic acid complex were less likely to be diagnosed with an abnormal Pap smear at 6 months [Odds ratio (OR): 0.39; 95% confidence interval (CI) 0.14-1.06] and 12 months (OR: 0.35; 95% CI 0.13-0.89), compared with the control group. After adjusting for confounding factors including age and smoking habit, this association remained significant. No effect was observed on HPV clearance or viral integration. The data from the current study suggest a protective effect of the ellagic acid complex on cervical cells, possibly through apoptosis, cell cycle arrest and repair mechanisms.

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Functional Screening in Human Cardiac Organoids Reveals a Metabolic Mechanism for Cardiomyocyte Cell Cycle Arrest

Background: The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of the adult. However, the key drivers of cardiomyocyte maturation and cell cycle arrest remain poorly defined. Methods: We developed a 96-well device for functional screening of human cardiac organoids to systematically screen for drivers of maturation and cell cycle arrest. Through interrogation of >10,000 cardiac organoids derived from human pluripotent stem cells, we systematically optimised parameters, including extracellular matrix, metabolic substrate and growth factor conditions that enhance cardiac tissue viability, reproducibility, function and maturation. Results: Under optimised maturation conditions, functional and molecular characterisation revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Additionally, under maturation conditions cardiomyocytes were refractory to mitogenic stimuli. We subsequently performed screening using a boutique library of kinase inhibitors and identified a small molecule capable of inducing cardiomyocyte cell cycle re-entry in mature human cardiac tissues and also in the adult mouse heart in vivo. Conclusions: These studies highlight that human organoids coupled with higher throughput screening platforms have the potential to rapidly expand our knowledge of cardiac biology and potentially unlock novel therapeutic strategies for cardiovascular disease.

Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of Paclitaxel and Curcumin for Brain Tumor Therapy.

Chemotherapy is one of the most important strategies for glioma treatment. However, the "impermeability" of the blood-brain barrier (BBB) impedes most chemotherapeutics from entering the brain, thereby rendering very few drugs suitable for glioma therapy, letting alone application of a combination of chemotherapeutics. Thereby, there is a pressing need to overcome the obstacles. A dual-targeting strategy was developed by a combination of magnetic guidance and transferrin receptor-binding peptide T7-mediated active targeting delivery. The T7-modified magnetic PLGA nanoparticle (NP) system was prepared with co-encapsulation of the hydrophobic magnetic nanoparticles and a combination of drugs (i.e., paclitaxel and curcumin) based on a "one-pot" process. The combined drugs yielded synergistic effects on inhibition of tumor growth via the mechanisms of apoptosis induction and cell cycle arrest, displaying significantly increased efficacy relative to the single use of each drug. Dual-targeting effects yielded a >10-fold increase in cellular uptake studies and a >5-fold enhancement in brain delivery compared to the nontargeting NPs. For the in vivo studies with an orthotopic glioma model, efficient brain accumulation was observed by using fluorescence imaging, synchrotron radiation X-ray imaging, and MRI. Furthermore, the antiglioma treatment efficacy of the delivery system was evaluated. With application of a magnetic field, this system exhibited enhanced treatment efficiency and reduced adverse effects. All mice bearing orthotopic glioma survived, compared to a 62.5% survival rate for the combination group receiving free drugs. This dual-targeting, co-delivery strategy provides a potential method for improving brain drug delivery and antiglioma treatment efficacy.

Nickel chloride (NiCl2) in hepatic toxicity: apoptosis, G2/M cell cycle arrest and inflammatory response.

Up to now, the precise mechanism of Ni toxicology is still indistinct. Our aim was to test the apoptosis, cell cycle arrest and inflammatory response mechanism induced by NiCl2 in the liver of broiler chickens. NiCl2 significantly increased hepatic apoptosis. NiCl2 activated mitochondria-mediated apoptotic pathway by decreasing Bcl-2, Bcl-xL, Mcl-1, and increasing Bax, Bak, caspase-3, caspase-9 and PARP mRNA expression. In the Fas-mediated apoptotic pathway, mRNA expression levels of Fas, FasL, caspase-8 were increased. Also, NiCl2 induced ER stress apoptotic pathway by increasing GRP78 and GRP94 mRNA expressions. The ER stress was activated through PERK, IRE1 and ATF6 pathways, which were characterized by increasing eIF2α, ATF4, IRE1, XBP1 and ATF6 mRNA expressions. And, NiCl2 arrested G2/M phase cell cycle by increasing p53, p21 and decreasing cdc2, cyclin B mRNA expressions. Simultaneously, NiCl2 increased TNF-α, IL-1β, IL-6, IL-8 mRNA expressions through NF-κB activation. In conclusion, NiCl2 induces apoptosis through mitochondria, Fas and ER stress-mediated apoptotic pathways and causes cell cycle G2/M phase arrest via p53-dependent pathway and generates inflammatory response by activating NF-κB pathway.

Abstract A10: A Cdk4-dependent phosphorylation threshold regulates the cell cycle entry decision

Abstract Cyclin-dependent kinases (Cdk's) are major cell cycle regulators and uncontrolled cell cycle entry is a hallmark of cancer. Cyclin D, the regulatory subunit of Cdk4, is misregulated in about 75% of human malignancies, leading to hyperactive Cdk4 and uncontrolled cell cycle entry. In sharp contrast, the total loss of Cyclin D-mediated activity results in permanent cell cycle arrest. Several functions of Cdk4 during cell cycle entry have been identified; however, the underlying mechanism for cell cycle arrest upon its inactivation remains unanswered. Here we employed CRISPR/Cas9 and rAAV-mediated gene editing to visualize Restriction Point passage in highly synchronized single cells and find that cyclin D:Cdk4/6 complexes perform a critical switch-like function that results in activation of cyclin E:Cdk2 complexes. Taken together, our data indicate that while cyclin E:Cdk2 is the rate-limiting step for transition across the Restriction Point, it is dependent on cyclin D:Cdk4/6 phosphorylation of an as of yet unknown molecule. Citation Format: Manuel Kaulich, Steven Dowdy. A Cdk4-dependent phosphorylation threshold regulates the cell cycle entry decision. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A10.

Abstract A27: Cell cycle control and drugs targeting the bioenergetics of cancer

Abstract Differences in responsiveness to chemotherapeutics among patients may be associated with multiple cell types that can be found in tumors and the circulation. Recurrent or refractory disease may result from natural selection of cells in response to therapy. To design new drugs and make better use of existing drugs differences between diseased and disease causing cells in response to a specific drug requires investigation. The Warburg Effect is characteristic of the majority of cancer cell types both in tumors and the circulation. Drugs that target the underlying biochemistry of essential bioenergetic processes unique to cancer have been shown to have activity against a broad scope of cell types and disease indications while maintaining a favorable safety profile. Recent studies have shown that CPI 613 may have concentration dependent differences in the nature of response, cell death or cell cycle arrest, when tested against H460, BXPC3 and SF539 cancer cells. Understanding the mechanisms of cell cycle arrest is an important factor in choosing other drugs to be used in combination to maximize patient benefit. Results evaluating drug effects on G2/M control proteins will be presented. Note: This abstract was not presented at the conference. Citation Format: Robert Shorr. Cell cycle control and drugs targeting the bioenergetics of cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A27.

Synthesis, characterization and anticancer effect of the ruthenium (II) polypyridyl complexes on HepG2 cells

As one of the major cell regulated center, mitochondria are closely associated with cell proliferation, apoptosis of tumor cell. In this work, four new ruthenium (II) polypyridyl complexes [Ru(bpy)2(FTTP)](ClO4)2 (1) (FTTP=11-(3-fluoro-naphthalen-2-yloxy)-4,5,9,14-tetraaza-benzo[b]triphenylene, bpy=2,2′-bipyridine), [Ru(phen)2(FTTP)](ClO4)2 (2) (phen=1,10-phenanthroline), [Ru(bpy)2(PTTP)](ClO4)2 (3) (PTTP=2-phenoxy-1,4,8,9-tetraazatriphenylene) and [Ru(phen)2(PTTP)](ClO4)2 (4) were synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. The cytotoxic activity, ability of inhibiting cell invasion, cell cycle arrest and apoptosis-inducing mechanism of these Ru(II) complexes have been investigated in detail by MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method, invasion assay, comet assay as well as western blotting techniques. Notably, complexes 1–4 displayed high cytotoxic activity against liver carcinoma HepG2 cells and the IC50 values of complexes 1–4 against HepG2 cells are 10.4±1.2, 9.3±0.6, 29.1±1.5 and 5.6±1.2μM, respectively. The comet assay showed that the complexes can induce DNA damage. The acridine orange (AO) and ethidium bromide (EB) staining method indicated that the complexes can cause apoptosis in HepG2 cells. Further studies showed that complexes 1–4 caused cell cycle arrest at G0/G1 phase and induced HepG2 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which involved an increase in the levels of reactive oxygen species (ROS), a decrease in the mitochondrial membrane potential, activation of caspases and Bcl-2 family proteins.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Dimethylfumarate inhibits melanoma cell proliferation via p21 and p53 induction and bcl-2 and cyclin B1 downregulation

Recent evidence suggests that dimethylfumarate (DMF), known as a highly potent anti-psoriatic agent, might have anti-tumorigenic properties in melanoma. It has recently been demonstrated that DMF inhibits melanoma proliferation by apoptosis and cell cycle inhibition and therefore inhibits melanoma metastasis. Nonetheless, the underlying mechanisms remain to be evaluated. To elucidate the effects of DMF on melanoma cell lines (A375, SK-Mel), we first performed cytotoxicity assays. No significant lactatedehydogenase (LDH) release could be found. In further analysis, we showed that DMF suppresses melanoma cell proliferation in a concentration-dependent manner. To examine whether these effects are conveyed by apoptotic mechanisms, we studied the amount of apoptotic nucleosomes and caspase 3/7 activity using ELISA analysis. Significant apoptosis was induced by DMF in both cell lines, and this could be paralleled with bcl-2 downregulation and PARP-1 cleavage. We also performed cell cycle analysis and found that DMF induced concentration-dependent arrests of G0/G1 as well as G2/M. To examine the underlying mechanisms of cell cycle arrest, we analyzed the expression profiles of important cell cycle regulator proteins such as p53, p21, cyclins A, B1, and D1, and CDKs 3, 4, and 6. Interestingly, DMF induced p53 and p21 yet inhibited cyclin B1 expression in a concentration-dependent manner. Other cell cycle regulators were not influenced by DMF. The knockdown of DMF induced p53 via siRNA led to significantly reduced apoptosis but had no influence on cell cycle arrest. We examined the adhesion of melanoma cells on lymphendothelial cells during DMF treatment and found a significant reduction in interaction. These data provide evidence that DMF inhibits melanoma proliferation by reinduction of important cell cycle inhibitors leading to a concentration-dependent G0/G1 or G2/M cell cycle arrest and induction of apoptosis via downregulation of bcl-2 and induction of p53 and PARP-1 cleavage. Hence, DMF might be an interesting agent in the treatment of melanoma and is worth further investigation in vivo.

Abstract LB-010: Maternal embryonic leucine zipper kinase (MELK): A novel target for radiosensitization that is independently prognostic in triple-negative breast cancers

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of RT, therefore approaches that result in radiosensitizaton in TNBC are critically needed. Our previous work identified one such novel molecular target and we sought to investigate the impact of MELK expression on radiation response and patient outcomes. Methods: MELK expression was measured with RNA-seq and RPPA in 2,061 breast tumor samples and 51 breast cancer cell (BCC) lines. Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays utilizing linearized pEYFP vector with FACS and qRT-PCR assessed NEHJ competency. We measured in vivo tumor growth and doubling time using xenografts in mice (16-20 tumors/group) treated with vehicle, RT, MELK-inhibitor, or combination. Kaplan-Meier analysis assessed the clinical impact of MELK on LR and OS. A Cox proportional hazards model was constructed to identify potential factors of LRF-survival in UVA and MVA. Results: MELK expression is absent in normal tissues and only expressed in cancerous tissue, suggesting a favorable therapeutic index. MELK expression was significantly elevated in human TNBC (p-value 7.5e-21) compared to non-TNBC. MELK expression was significantly correlated with radioresistance (R: 0.62, p-value 0.003). Inhibition of MELK using both siRNA and a small molecule inhibitor induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.5-1.6 through impaired dsDNA repair and cell cycle arrest mechanisms. MELK protein interacts with Ku70 and Ku80 to mediate NHEJ which is significantly impaired at 1, 3, and 6 hours after MELK inhibitor treatment (p-value<0.01). MELK inhibition either with gene knock-down or small molecule inhibitor significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor doubling time and tumor growth (median tumor doubling 7.95 vs. 29.1 days for RT vs. MELK inhibition+RT, p-value <0.0001). Breast cancer patients whose tumors have high expression of MELK had markedly higher rates of LR after RT and an overall poorer prognosis than patients with low expression of MELK (HR for LR 1.89-2.23, p-value<0.001; HR for OS 1.46-3.3; p-value<0.001 in 3 independent datasets). In multivariable analysis only MELK expression and grade were significantly associated with worse LRF survival (HR:1.35,p-value<0.01). Conclusion: Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target to increase radiosensitivity in TNBC. Citation Format: Corey Speers, Alyssa Santola, Shuang G. Zhao, Meilan Liu, Vishal Kothari, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Lori J. Pierce, Felix Y. Fang. Maternal embryonic leucine zipper kinase (MELK): A novel target for radiosensitization that is independently prognostic in triple-negative breast cancers. [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 LB-010.

Pooled screening for antiproliferative inhibitors of protein-protein interactions.

Protein-protein interactions (PPIs) are emerging as a promising new class of drug targets. Here, we present a novel high-throughput approach to screen inhibitors of PPIs in cells. We designed a library of 50,000 human peptide binding motifs and used a pooled lentiviral system to express them intracellularly and screen for their effects on cell proliferation. We thereby identified inhibitors that drastically reduced the viability of a pancreas cancer line (RWP1) while leaving a control line virtually unaffected. We identified their target interactions computationally, and validated a subset in experiments. We also discovered their potential mechanisms of action including apoptosis and cell cycle arrest. Finally, we confirmed that synthetic lipopeptide versions of our inhibitors have similarly specific and dosage dependent effects on cancer cell growth. Our screen reveals new drug targets and peptide drug leads and it provides a rich dataset covering phenotypes for inhibition of thousands of interactions.

Chronic DNA Replication Stress Reduces Replicative Lifespan of Cells by TRP53-Dependent, microRNA-Assisted MCM2-7 Downregulation.

Circumstances that compromise efficient DNA replication, such as disruptions to replication fork progression, cause a state known as DNA replication stress (RS). Whereas normally proliferating cells experience low levels of RS, excessive RS from intrinsic or extrinsic sources can trigger cell cycle arrest and senescence. Here, we report that a key driver of RS-induced senescence is active downregulation of the Minichromosome Maintenance 2–7 (MCM2-7) factors that are essential for replication origin licensing and which constitute the replicative helicase core. Proliferating cells produce high levels of MCM2-7 that enable formation of dormant origins that can be activated in response to acute, experimentally-induced RS. However, little is known about how physiological RS levels impact MCM2-7 regulation. We found that chronic exposure of primary mouse embryonic fibroblasts (MEFs) to either genetically-encoded or environmentally-induced RS triggered gradual MCM2-7 repression, followed by inhibition of replication and senescence that could be accelerated by MCM hemizygosity. The MCM2-7 reduction in response to RS is TRP53-dependent, and involves a group of Trp53-dependent miRNAs, including the miR-34 family, that repress MCM expression in replication-stressed cells before they undergo terminal cell cycle arrest. miR-34 ablation partially rescued MCM2-7 downregulation and genomic instability in mice with endogenous RS. Together, these data demonstrate that active MCM2-7 repression is a physiologically important mechanism for RS-induced cell cycle arrest and genome maintenance on an organismal level.

The studies on the cytotoxicity in vitro, cellular uptake, cell cycle arrest and apoptosis-inducing properties of ruthenium methylimidazole complex [Ru(MeIm)4(p-cpip)]2 +

A new ruthenium methylimidazole complex [Ru(MeIm)4(p-cpip)]2+ (Ru1, p-cpip=2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, MeIm=1-methylimidazole) has been synthesized and characterized. The cellular uptake, in vitro cytotoxicities, cell cycle arrest and apoptosis-inducing mechanism of this Ru(II) complex have been extensively explored by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, Comet assay, inverted fluorescence microscope as well as Western blotting experimental techniques. Notably, Ru1 displayed relatively high cytotoxic activity against lung cancer A549 cells and had high selectivity between tumor and normal cells in comparison with cisplatin. Further studies showed that Ru1 caused cell cycle arrest at G0/G1 phase and induced apoptosis via the mitochondrial pathway, which involved reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and Bcl-2 and caspase correlative family member activation. For providing more information about the possible antitumor mechanism, the in vitro DNA binding studies have been also investigated by different spectrophotometric methods, thermal denaturation and viscosity measurements.

Cyproheptadine exhibits antitumor activity in urothelial carcinoma cells by targeting GSK3β to suppress mTOR and β-catenin signaling pathways

Cyproheptadine, a serotonin antagonist, has recently been reported to function as a novel therapeutic agent by inhibiting PI3K/AKT signaling in several human cancers. However, the therapeutic effect of cyproheptadine in urothelial carcinoma (UC) has never been explored. In this study, we determined the effect of cyproheptadine on the growth of five human UC cell lines and an in vivo xenograft model. The results showed that cyproheptadine exerted an inhibitory effect on the proliferation of UC cells both in vitro and in vivo. Cyproheptadine also induced cell cycle arrest in the G1 phase, subsequently followed by apoptosis and necrosis. The underlying mechanisms of cell cycle arrest were associated with the reduction of c-Myc, induction of p21 and p27, and the stabilization of Rb expression. In addition, the suppression of the GSK3β/TSC2/mTOR pathway and deregulation of the GSK3β/β-catenin signaling were observed in cyproheptadine-treated UC cells. Furthermore, cyproheptadine-induced apoptosis was associated with ANGPTL4 expression followed by activation of caspase3 and PARP in UC cells. Our experimental results provide evidence that cyproheptadine is a suitable therapeutic agent for the treatment of UC.

Abstract 82: Analysis of the role of MDM2 in regulation of cell cycle arrest through p21 pathways in LNCaP-MST cells using PCR array

Abstract Cell cycle checkpoints are imperative for assuring genomic fidelity and integrity during cell division. In normal cells, the cell cycle arrest occurs in response to nuclear DNA damage via activation and inactivation of a series of cell cycle regulatory molecules that can impact the transition from G1 to S phase and G2 to M phase. However, in cancerous cells, this process is disrupted resulting in uncontrolled cell proliferation and resistance to apoptosis. Therefore, cell cycle regulatory molecules are accepted as suitable targets for drug development and cancer treatment. It has been well known that overexpression of MDM2, a negative regulator of p53, will eventually lead to the inactivation of cell cycle control and loss of apoptotic ability in many tumors. In this study, we aimed to investigate the effectiveness of MDM2 inhibition in mediating cell cycle arrest through p53 dependent pathways by using the MDM2 transfected prostate cancer cells (LNCaP-MST). During our experiments the LNCaP-MST cells were treated for 24 hrs with 20 uM Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction. The impact of Nutlin-3 treatment on the gene expression profile of LNCaP-MST cells was established by using the cell cycle pathway specific PCR array. Our study clearly demonstrates a significant increase of p21 gene expression after Nutlin-3 treatment, which indicates a possible restoration and release of the transcriptional activity of p53 in LNCaP-MST cells. In addition to the elevation of p21, a significant increase in the expression of Cyclin-Dependent Kinase 4 Inhibitor B (CDKN2B) along with multiple folds increase in the expression of Growth Arrest and DNA Damage 45 (GADD45A) genes were also found. These results clearly point towards efficient initiation of cell cycle arrest mechanisms in LNCaP-MST cells, possibly through p21 and CDKN2B mediated inhibition of the CDKs (cyclin dependent kinases). In addition, it appears that MDM2 inhibition may effectively block both G1 to S and G2 to M transition and cause DNA damage through apoptosis that could lead to the death of LNCaP-MST cells. Activation of the above outlined mechanisms following MDM2 inhibition is quite evident due to increased expression of GADD45A and decreased levels of anti-apoptotic B-cell lymphoma 2 (Bcl-2) genes. Our results offer convincing evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest and apoptosis during cancer treatment. (This research was supported by the generous funds provided by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Khalid Alhazzani, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu. Analysis of the role of MDM2 in regulation of cell cycle arrest through p21 pathways in LNCaP-MST cells using PCR array. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 82. doi:10.1158/1538-7445.AM2015-82

Abstract A49: Inhibition of mTOR induces chemoresistance in B-cell acute lymphoblastic leukemia

Abstract B-ALL is the most prominent cancer in pediatric patients. Most patients are responsive to the current chemotherapy, but B-ALLs with activated oncogenic kinases including BCR-ABL, PDGFR and JAK have a high risk of relapse. Currently, patients with the BCR-ABL translocation are successfully treated when the BCL-ABL inhibitor dasatinib is added to their chemotherapy regimen. In accord, we found that dasatinib sensitizes B-ALL cell lines to drugs used during induction and consolidation phase of chemotherapy. However, dasatinib protects BCR-ABL leukemia cell lines from killing by maintenance phase drugs methotrexate and 6-mercaptopurine. This protection only occurs in cells where dasatinib reduces downstream mTOR activity. To directly test if reduced mTOR activity can account for the protection, we assessed the effects of the allosteric mTOR inhibitor rapamycin and active-site mTOR inhibitor MLN0128. Indeed, both mTOR inhibitors consistently protect B-ALL cells from killing by methotrexate and 6-mercaptopurine. Inhibitors of AKT and PI3K also result in varying degrees of chemoprotection, which correlates strongly with the degree to which they downregulate mTOR activity. The chemoprotective effect of mTOR inhibition correlates with cell cycle arrest but other mechanisms may also be involved. These data suggest that mTOR inhibition is detrimental in some chemotherapeutic combinations, whether by inhibitors directly targeting mTOR or indirectly targeting upstream effectors. The identification of such drug combination effects is an important consideration for the current use of dasatinib and future use of PI3K/AKT/mTOR inhibitors. Citation Format: Thanh-Trang Vo, Duc Nguyen, Brandon Lui, Mengrou Lu, Sharmila Mallya, David Fruman. Inhibition of mTOR induces chemoresistance in B-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A49.