Cell Cycle Regulatory Proteins Research Articles

CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

Tat-NTS Suppresses the Proliferation, Migration and Invasion of Glioblastoma Cells by Inhibiting Annexin-A1 Nuclear Translocation.

Prevention of the nuclear translocation of ANXA1 with Tat-NTS was recently reported to alleviate neuronal injury and protect against cerebral stroke. However, the role that Tat-NTS plays in the occurrence and development of gliomas still needs to be elucidated. Therefore, human glioblastoma (GB) cells were treated with various concentrations of Tat-NTS for 24h, and cell proliferation, migration and invasion were assessed with CCK-8 and Transwell assays. The nuclear translocation of ANXA1 was evaluated by subcellular extraction and immunofluorescence, and protein expression levels were detected by Western blot analysis. In addition, the activity of MMP-2/9 was measured by gelatin zymography. The results revealed that Tat-NTS significantly inhibited the nuclear translocation of ANXA1 in U87 cells and inhibited the proliferation, migration and invasion of GB cells. Tat-NTS also suppressed cell cycle regulatory proteins and MMP-2/-9 activity and expression. Moreover, Tat-NTS reduced the level of p-p65 NF-κB in U87 cells. These results suggest that the Tat-NTS-induced inhibition of GB cell proliferation, migration and invasion is closely associated with the induction of cell cycle arrest, downregulation of MMP-2/-9 expression and activity and suppression of the NF-κB signaling pathway. Thus, Tat-NTS may be a potential chemotherapeutic agent for the treatment of GB.

Pharmacological Activation of Autophagy Restores Cellular Homeostasis in Ultraviolet-(B)-Induced Skin Photodamage.

Ultraviolet (UV) exposure to the skin causes photo-damage and acts as the primary etiological agent in photo-carcinogenesis. UV-B exposure induces cellular damage and is the major factor challenging skin homeostasis. Autophagy allows the fundamental adaptation of cells to metabolic and oxidative stress. Cellular dysfunction has been observed in aged tissues and in toxic insults to cells undergoing stress. Conversely, promising anti-aging strategies aimed at inhibiting the mTOR pathway have been found to significantly improve the aging-related disorders. Recently, autophagy has been found to positively regulate skin homeostasis by enhancing DNA damage recognition. Here, we investigated the geno-protective roles of autophagy in UV-B-exposed primary human dermal fibroblasts (HDFs). We found that UV-B irradiation to HDFs impairs the autophagy response in a time- and intensity-independent manner. However, improving autophagy levels in HDFs with pharmacological activators regulates the UV-B-induced cellular stress by decreasing the induction of DNA photo-adducts, promoting the DNA repair process, alleviating oxidative and ER stress responses, and regulating the expression levels of key cell cycle regulatory proteins. Autophagy also prevents HDFs from UV-B-induced nuclear damage as is evident in TUNEL assay and Acridine Orange/Ethidium Bromide co-staining. Salubrinal (an eIF2α phosphatase inhibitor) relieves ER stress response in cells and also significantly alleviates DNA damage and promotes the repair process in UV-B-exposed HDFs. P62-silenced HDFs show enhanced DNA damage response and also disturb the tumor suppressor PTEN/pAKT signaling axis in UV-B-exposed HDFs whereas Atg7-silenced HDFs reveal an unexpected consequence by decreasing the UV-B-induced DNA damage. Taken together, these results suggest that interventional autophagy offers significant protection against UV-B radiation-induced photo-damage and holds great promise in devising it as a suitable therapeutic strategy against skin pathological disorders.

Hesperidin and its aglycone hesperetin in breast cancer therapy: A review of recent developments and future prospects.

Breast cancer (BC) has high incidence and mortality rates, making it a major global health issue. BC treatment has been challenging due to the presence of drug resistance and the limited availability of therapeutic options for triple-negative and metastatic BC, thereby urging the exploration of more effective anti-cancer agents. Hesperidin and its aglycone hesperetin, two flavonoids from citrus species, have been extensively evaluated for their anti-cancer potentials. In this review, available literatures on the chemotherapeutic and chemosensitising activities of hesperidin and hesperetin in preclinical BC models are reported. The safety and bioavailability of hesperidin and hesperetin as well as the strategies to enhance their bioavailability are also discussed. Overall, hesperidin and hesperetin can inhibit cell proliferation, migration and BC stem cells as well as induce apoptosis and cell cycle arrest in vitro. They can also inhibit tumour growth, metastasis and neoplastic changes in tissue architecture in vivo. Moreover, the co-administration of hesperidin or hesperetin with doxorubicin, letrozole or tamoxifen can enhance the efficacies of these clinically available agents. These chemotherapeutic and chemosensitising activities of hesperidin and hesperetin have been linked to several mechanisms, including the modulation of signalling pathways, glucose uptake, enzymes, miRNA expression, oxidative status, cell cycle regulatory proteins, tumour suppressor p53, plasma and liver lipid profiles as well as DNA repair mechanisms. However, poor water solubility, extensive phase II metabolism and apical efflux have posed limitations to the bioavailability of hesperidin and hesperetin. Various strategies for bioavailability enhancement have been studied, including the utilisation of nano-based drug delivery systems and the co-administration of hesperetin with other flavonoids. In particular, nanoformulated hesperidin and hesperetin possess greater chemotherapeutic and chemosensitising activities than free compounds. Despite promising preclinical results, further safety and efficacy evaluation of hesperidin and hesperetin as well as their nanoformulations in clinical trials is required to ascertain their potentials to be developed as clinically useful agents for BC treatment.

Evaluation of anticancer effects of Juniperus communis extract on hepatocellular carcinoma cells in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and accounts for the fourth leading cause of all cancer deaths. Scientific evidence has found that plant extracts seem to be a reliable choice due to their multitarget effects against HCC. Juniperus communis has been used for centuries in traditional medicine and its anticancer properties have been reported. As a result, the purpose of the study was to investigate the anticancer effect and mechanism of J. communis extract (JCo extract) on HCC in vitro and in vivo. In the present study, we found that JCo extract inhibited the growth of human HCC cells by inducing cell cycle arrest at the G0/G1 phase, extensive apoptosis and suppressing metastatic protein expressions in HCC cells. Moreover, the combinational treatment of JCo and VP-16 was found to enhance the anticancer effect, revealing that JCo extract might have the potential to be utilized as an adjuvant to promote HCC treatment. Furthermore, in vivo study, JCo extract significantly suppressed HCC tumor growth and extended the lifespan with no or low systemic and pathological toxicity. JCo extract significantly up-regulated the expression of pro-apoptotic proteins and tumor suppressor p53, suppressed VEGF/VEGFR autocrine signaling, down-regulated cell cycle regulatory proteins and MMP2/MMP9 proteins. Overall, our results provide a basis for exploiting JCo extract as a potential anticancer agent against HCC.

Surfactin induces autophagy, apoptosis, and cell cycle arrest in human oral squamous cell carcinoma.

To investigate the anticancer effects and underlying mechanisms of surfactin on human oral squamous cell carcinoma (OSCC). The capacity of surfactin to induce apoptosis, autophagy, and cell cycle arrest of two different human OSCC cell lines was investigated by cell viability, acridine orange staining, and cell cycle regulatory protein expression, respectively. The signaling network underlying these processes were determined by the analysis of reactive oxygen species (ROS) generation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, endoplasmic reticulum (ER) stress-related protein levels, calcium release, mitogen-activated protein kinases activation, and cell cycle regulatory protein expression through corresponding reagents and experiments under various experimental conditions using specific pharmaceutical inhibitors or small interfering RNAs. Surfactin was able to induce apoptosis through NADPH oxidase/ROS/ER stress/calcium-downregulated extracellular signal-regulated kinases 1/2 pathway. Surfactin could also lead to autophagy that shared the common regulatory signals with apoptosis pathway until calcium node. Cell cycle arrest at G2 /M phase caused by surfactin was demonstrated through p53 and p21 accumulation combined p34cdc2 , phosphorylated p34cdc2 , and cyclin B1 inhibition, which was regulated by NADPH oxidase-derived ROS. Surfactin could induce apoptosis, autophagy, and cell cycle arrest in ROS-dependent manner, suggesting a multifaced anticancer agent for OSCC.

Esomeprazole enhances the effect of ionizing radiation to improve tumor control.

The resistance of cancer cells to radiation-based treatment is a major clinical challenge confounding standard of care in cancer. This problem is particularly notable in many solid tumors where cancer cells are only partially responsive to radiation therapy. Combination of radiation with radiosensitizers is able to enhance tumor cell killing. However, currently available radiosensitizers are associated with significant normal tissue toxicity. Accordingly, there is an unmet need to develop safer and more effective radiosensitizers to improve tumor control. Here, we evaluated the radiosensitizing effect of the FDA-approved drug esomeprazole in normal and radioresistant human head and neck squamous cell carcinoma (HNSCC) cells in vitro, and in a mouse model of HNSCC. For the in vitro studies, we used cancer cell colony formation (clonogenicity) assay to compare cancer cell growth in the absence or presence of esomeprazole. To determine mechanism(s) of action, we assessed cell proliferation and profiled cell cycle regulatory proteins. In addition, we performed reverse phase protein array (RPPA) study to understand the global effect of esomeprazole on over 200 cancer-related proteins. For the in vivo study, we engrafted HNSCC in a mouse model and compared tumor growth in animals treated with radiation, esomeprazole, and combination of radiation with esomeprazole. We found that esomeprazole inhibits tumor growth and dose-dependently enhances the cell killing effect of ionizing radiation in wildtype and p53-mutant radioresistant cancer cells. Mechanistic studies demonstrate that esomeprazole arrests cancer cells in the G1 phase of the cell cycle through upregulation of p21 protein and inhibition of cyclin-dependent kinases (Cdks) type 1 (Cdk1) and type 2 (Cdk2). In vivo data showed greater tumor control in animals treated with combination of radiation and esomeprazole compared to either treatment alone, and that this was associated with inhibition of cell proliferation in vivo. In addition, combination of esomeprazole with radiation significantly impaired repair following radiation-induced DNA damage. Our studies indicate that esomeprazole sensitizes cancer cells to ionizing radiation, and is associated with upregulation of p21 to arrest cells in the G1 phase of the cell cycle. Our findings have significant therapeutic implications for the repurposing of esomeprazole as a radiosensitizer in HNSCC and other solid tumors.

Abstract 1958: Inhibition of CDK4/6 in head and neck squamous cell carcinoma dismantles key DNA repair pathways in response to radiation treatment

Abstract Introduction: Conventional treatment for head and neck squamous cell carcinoma (HNSCC) relies on surgery and radiation for cure; locoregional failure is often related to recurrence after radiation. CDKN2A, coding for the CDK4/6 inhibitory protein, p16INK4a (p16), is among the most commonly lost tumor suppressors in HNSCC. Palbociclib, a chemotherapeutic agent that is FDA-approved for breast cancer, is a selective CDK4/6 inhibitor. FOXM1 is a key cell cycle regulatory protein whose activation is controlled by CDK4/6. FOXM1 controls cell proliferation, DNA damage repair and suppresses senescence. The current study examines the role of CDK4/6 and its targets in response to radiation in HNSCC. We demonstrate inhibiting CDK4/6 can synergize with radiation, partially via suppression of FOXM1 signaling and impact on senescence. Experimental Procedure: Our study focuses on utilizing palbociclib to increase radiation response in HPV(-) oral cavity HNSCC and understanding mechanisms of potential radiosensitization. We examined cell viability after concurrent palbociclib and radiation (P+RT) application using proliferation assays in 2-dimensional (2D) and 3D organoid models of immortalized cell lines (HN5 and Cal27), and calculated synergy by Compusyn analysis. After 3-days treatment, we performed western blot to evaluate DNA damage response (DDR) proteins and β-galactosidase (β -gal) staining to evaluate the senescent phenotype. Flow cytometry studies were used to measure cell cycle arrest and QPCR to measure expression of cell cycle regulators. Results: P+RT (1µM palbociclib + 2 or 4Gy RT) demonstrated consistent synergy in both 2D and 3D viability assays. Comparing P+RT to RT alone, flow cytometry data revealed a 2-fold increase in the G1 arrested population, and DNA damage (measured by γ-H2AX level) increased by 2.3 fold. Additionally, Rad51 and Ku80 showed a 2 and 10-fold decrease respectively, suggesting an impact on DDR repair via both the homologous recombination and non-homologous end joining pathways. On transient knockdown of CDK-4 or 6, or both, we observed that senescence was the prevailing response - concurrent inhibition of CDK4 and CDK6 was the most profound, and levels of senescence based on β -gal staining results matched that observed with palbociclib treatment. P+RT demonstrated a robust 3-fold decrease in FOXM1 mRNA level and a 10-fold decrease in protein expression. We observed a 5-fold decrease in FOXM1 levels with CDK6 inhibition compared to a 2-fold decrease with CDK4 inhibition alone. Independently, FOXM1 inhibition induced apoptosis in HNSCC to a level higher than staurosporine-control treated cells. Conclusions: Our results suggest that palbociclib may make HNSCC more susceptible to RT via the induction of senescence related to suppressed signaling through FOXM1.We propose P+RT as a rational and potentially effective treatment strategy in HNSCC Citation Format: Nitisha Shrivastava, Carlos Thomas, Daniel Li, Cory D. Fulcher, Michael B. Prystowsky, Indranil Basu, Chandan Guha, Thomas J. Ow. Inhibition of CDK4/6 in head and neck squamous cell carcinoma dismantles key DNA repair pathways in response to radiation treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1958.

Abstract 1963: High expression of Cyclin D1 is an independent marker for favorable prognosis in Middle Eastern breast cancer

Abstract Cyclin D1 protein regulates cell cycle progression which is mediated by its interactions with cyclin-dependent kinases. Over-expression of Cyclin D1 has been observed in several human cancers. This study was conducted to evaluate Cyclin D1 expression in a large cohort of Middle Eastern breast cancers and determine its prognostic significance. Cyclin D1 expression was assessed immunohistochemically and its association with clinico-pathological parameters was analyzed. Cyclin D1 was over-expressed in 59.4% (596/1003) of cases and significantly associated with a subset of breast cancers having favorable prognostic features such as low grade (p < 0.0001), low stage (p = 0.0276), estrogen receptor positive (p < 0.0001) and progesterone receptor positive (p < 0.0001) tumors. An inverse association was found with triple negative breast cancers (p < 0.0001). More importantly, Cyclin D1 expression was an independent predictor of favorable overall survival in our cohort (Hazard ratio = 0.69; 955 confidence interval = 0.48 - 0.98; p = 0.0405). Also, tumors that highly expressed cyclin D1 had a longer recurrence free survival. However, recurrence free survival was only significant in univariate analysis. In conclusion, our results reinforced the role of cyclin D1 in breast cancer pathology and revealed its expression as a valuable independent prognostic indicator for breast cancer from Middle Eastern ethnicity. Citation Format: Khawla S. Al-Kuraya, Abdul K. Siraj, Sandeep K. Parvathareddy, Sarah Siraj, Saud Azam, Padmanaban Annaiyappanaidu, Maria Angelita Sabido, Mark Ranier Diaz. High expression of Cyclin D1 is an independent marker for favorable prognosis in Middle Eastern breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1963.

Abstract 1252: The mechanism of the drug, SHetA2, in cervical cancer cells involves growth inhibition, mitochondria damage and release of AIF to cause caspase-independent cell death

Abstract Introduction SHetA2 is an orally-bioavailable, novel small-molecule drug that is entering first in human trials for gynecologic cancers. Upon binding to three related heat shock protein A chaperones (HSPA 5, 8 and 9 genes encoding BiP, hsc70 and mortalin proteins, respectively), SHetA2 causes release of their client proteins and inhibition of mortalin import into mitochondria. The purpose of this study was to evaluate the potential of SHetA2 for cervical cancer therapy and to explore its mechanism of action. Methods: The cytotoxic effect of SHetA2 on cervical cancer cell lines, C33a, C-4 II, CaSki, ME-180 and SiHa was assessed using a MTT metabolic viability assay. The effects of SHetA2 on cell cycle arrest were evaluated by flow cytometric analysis of PI staining and western blots. SHetA2 effects on anchorage-independent growth and cell migration were analyzed using soft-agar colony formation and wound healing scratch assays, respectively. The effect of SHetA2 on mitochondria was assessed using the JC-1 dye, measurement of ATP, electron microscopy, confocal microscopy and Seahorse XF Cell Mito Stress. Result: SHetA2 significantly reduced proliferation of all cervical cell lines in time- and dose- dependent manners. This drug also inhibited anchorage-independent growth, cell migration and cell cycle progression through G1-phase cell-cycle arrest in C-33 A and CaSki, and G2- arrest in SiHa, in association with relevant alterations of cell-cycle regulatory proteins. SHetA2 significantly decreased mitochondrial membrane potential and ATP production indicating mitochondrial toxicity. Electron microscopy demonstrated mitochondrial swelling and increased autophagic vesicles in SHetA2-treated SiHa cells. Confocal microscopy using mitochondria-specific dye demonstrated loss of mitochondrial networking and structural damage. Consistent with this, our western blot results demonstrated significant downregulation in the expression of proteins involved in mitochondrial fusion indicating that SHetA2 causes inhibition of mitochondrial fusion. Metabolic studies demonstrated that SHetA2 caused attenuation of efficient oxidative phosphorylation, ATP production, and elevation of basal glycolysis. Eventual SHetA2-induced cell death was not affected by a caspase inhibitor. Further mechanistic study demonstrated translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, along with nuclear DNA damage. Conclusions: SHetA2 inhibits growth, migration and invasion of cervical cancer cell lines in association with cell cycle arrest and mitochondrial toxicity. SHetA2-induced mitochondrial damage in cervical cancer cell lines likely mediates release of AIF to cause cell death in a caspase-independent manner. Citation Format: Rajani Rai, Vishal Chandra, Doris M. Benbrook. The mechanism of the drug, SHetA2, in cervical cancer cells involves growth inhibition, mitochondria damage and release of AIF to cause caspase-independent cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1252.

HMT Exerts an Anticancer Effect by Targeting PAK-1

Heamatang (HMT) is a classic medicinal formula used in traditional Chinese and Korean medicine; it contains seven distinct components, mainly of herbal origin. HMT is used as an antiaging remedy, treating urinary disorders and increasing energy and vitality. However, the therapeutic applications of this formula have not been evaluated with evidence-based science. Therefore, we assessed HMT through various in vitro methods, including cell viability assay, fluorescence-activated cell sorting assay (FACS), Western blotting, migration assay, three-dimensional (3D) cell culture, siRNA-mediated PAK-1 knockdown, and crystal violet assays. HMT decreased PAK-1 expression in PC-3 cells and inhibited cell viability, growth, and motility. The inhibition of cell motility by HMT was correlated with PAK-1-mediated inhibition of Lim domain kinase (LIMK) and cofilin. HMT induced G1 arrest and apoptosis through the transcriptional regulation of cell cycle regulatory proteins and apoptosis-related proteins (increase in c-cas3 and inhibition of PARP and BCL-2). Moreover, HMT suppressed PAK-1 expression, leading to the inhibition of AKT activities. Finally, we showed that decursin was the active ingredient involved in the inhibitory effect of HMT on PAK-1. Our findings demonstrated that HMT exerts its anticancer influence through the inhibition of PAK-1. The HMT formula could be applied in various fields, including functional health food and pharmaceutical development.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth.

Resveratrol (RSV) is a natural compound that displays several pharmacological properties, including anti-cancer actions. However, its clinical application is limited because of its low solubility and bioavailability. Here, the antiproliferative and anti-inflammatory activity of a series of phenylacetamide RSV derivatives has been evaluated in several cancer cell lines. These derivatives contain a monosubstituted aromatic ring that could mimic the RSV phenolic nucleus and a longer flexible chain that could confer a better stability and bioavailability than RSV. Using MTT assay, we demonstrated that most derivatives exerted antiproliferative effects in almost all of the cancer cell lines tested. Among them, derivative 2, that showed greater bioavailability than RSV, was the most active, particularly against estrogen receptor positive (ER+) MCF7 and estrogen receptor negative (ER-) MDA-MB231 breast cancer cell lines. Moreover, we demonstrated that these derivatives, particularly derivative 2, were able to inhibit NO and ROS synthesis and PGE2 secretion in lipopolysaccharide (LPS)-activated U937 human monocytic cells (derived from a histiocytoma). In order to define the molecular mechanisms underlying the antiproliferative effects of derivative 2, we found that it determined cell cycle arrest at the G1 phase, modified the expression of cell cycle regulatory proteins, and ultimately triggered apoptotic cell death in both breast cancer cell lines. Taken together, these results highlight the studied RSV derivatives, particularly derivative 2, as promising tools for the development of new and more bioavailable derivatives useful in the treatment of breast cancer.

C646 inhibits G2/M cell cycle-related proteins and potentiates anti-tumor effects in pancreatic cancer

The activity of histone acetyltransferases (HATs) plays a central role in an epigenetic modification in cooperation with HDACs (histone deacetyl transferases). It is likely that malfunction of this enzymatic machinery controlling epigenetic modification is relevant to carcinogenesis and tumor progression. However, in pancreatic cancer, the clinical relevance of HAT activity and histone acetylation has remained unclear. We identified that H3 acetylation was expressed in all pancreatic cancer patients, indicating that H3 acetylation may be essential in pancreatic cancer cells. We also found that the HAT inhibitor C646 augmented anti-tumor effects in vitro by inhibiting cell proliferation and cell cycle progression concomitantly with suppression of acetylated H3K9 and H3K27 expression. C646 or p300 and CBP (CREB-binding protein)-specific siRNA treatment inhibited the transcription of the G2/M cell cycle regulatory proteins cyclin B1 and CDK1 (cyclin-dependent kinase 1). C646 treatment also inhibited tumor growth in vivo in a xenograft mouse model. C646 could be an effective therapeutic agent for pancreatic cancer. The epigenetic status of pancreatic cancers based on their level of histone H3 acetylation may influence patient survival. Epigenetic stratification according to H3K27 acetylation could be useful for predicting disease prognosis as well as the therapeutic efficacy of C646 in pancreatic cancer.

Cell Cycle Regulatory Protein Expression in Multinucleated Giant Cells of Giant Cell Tumor of Bone: do They Proliferate?

Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15INK4b, p16INK4a, p27KIP1, p53 through its effector p21WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.

Pogostemon cablin extract as an anticancer agent on human acute myeloid leukemia.

Pogostemon cablin has been indicated to treat many kinds of diseases and the progression of cancers, such as colorectal cancer. However, the effects of P. cablin extract (PPa extract) against acute myeloid leukemia have not been investigated. Thus, this study explored the anticancer potential of PPa extract and its mechanism in HL‐60 cells. The MTT assay results showed that PPa extract significantly inhibited the proliferation of HL‐60 cells in a dose‐dependent manner and affected cell morphology, causing cell shrinkage and the formation of debris. PPa extract blocked cell cycle progression at the G0/G1 phase in a dose‐ and time‐dependent manner and induced cell apoptosis, as shown by the observation of DNA fragments and apoptotic bodies. Furthermore, PPa extract caused the accumulation of a population of cells at G0/G1 phase via a reduction in p‐Rb, increasing p21 expression, and downregulating cell cycle regulator protein expression. Then, PPa extract was found to activate the extrinsic and intrinsic apoptosis pathways, leading to cell death. These data demonstrated that PPa extract exerted inhibitory activity and triggered cell apoptosis in HL‐60 cells and that PPa extract might be a chemopreventive agent for cancer therapy.

High Expression of Cyclin D1 is an Independent Marker for Favorable Prognosis in Middle Eastern Breast Cancer.

PurposeThe cyclin D1 protein regulates cell cycle progression which is mediated by its interactions with cyclin-dependent kinases. Over-expression of cyclin D1 has been observed in several human cancers. This study was conducted to evaluate cyclin D1 expression in a large cohort of Middle Eastern breast cancers and determine its prognostic significance.Patients and MethodsCyclin D1 expression was assessed immunohistochemically and its association with clinico-pathological parameters was analyzed in 1003 breast cancer patients.ResultsCyclin D1 was over-expressed in 59.4% (596/1003) of cases and significantly associated with a subset of breast cancers having favorable prognostic features, such as low grade (p < 0.0001), low stage (p = 0.0276), estrogen receptor (p < 0.0001) and progesterone receptor positive (p < 0.0001) tumors. An inverse association was found with triple negative breast cancers (p < 0.0001). More importantly, cyclin D1 expression was an independent predictor of favorable overall survival in our cohort (hazard ratio = 0.70; 95% confidence interval = 0.50–0.98; p = 0.0395). Also, tumors that highly expressed cyclin D1 had a longer recurrence-free survival. However, this significant association was seen only in univariate analysis. We also found cyclin D1 to be associated with phospho-Rb in luminal subtype of breast cancer and co-expression of both these markers was an independent predictor of luminal A breast cancer.ConclusionOur results reinforced the role of cyclin D1 in breast cancer pathology and revealed its expression as a valuable independent prognostic indicator for breast cancer from Middle Eastern ethnicity.

Theabrownin Induces Cell Apoptosis and Cell Cycle Arrest of Oligodendroglioma and Astrocytoma in Different Pathways.

Theabrownin (TB), a natural compound present in the fresh leaves of green tea, is a potential antitumor agent. However, so far whether and how TB affects glioma is unclear. In this study, we investigated the effect of TB on astroglioma and oligodendroglioma cells. Surprisingly, TB significantly reduced the viabilities of HOG and U251 cells in a dose-dependent manner, which was accompanied by the upregulation of active-Casp-3, Bax, and PTEN; meanwhile, the antiapoptotic gene Bcl-2 was downregulated. In addition, TB treatment induced cell cycle arrest at the G1 and G2/M phases in HOG and U251 cells, respectively. TB treatment caused the downregulating of c-myc, cyclin D, CDK2, and CDK4 and upregulating of p21 and p27 in the HOG cell, while TB increased P53, p21, and p27 levels and decreased the levels of cell cycle regulator proteins such as CDK and cyclin A/B in the U251 cells. Therefore, the c-myc- and P53-related mechanisms were proposed for cell cycle arrest in these two glioma cell lines, respectively. Overall, our findings indicated that TB could be a novel candidate drug for the treatment of gliomas.

Fumonisin B1 triggers carcinogenesis via HDAC/PI3K/Akt signalling pathway in human esophageal epithelial cells

Fumonisin B1 (FB1) is a contaminant that commonly present in the global environment, especially in food and feed. Epidemiologic studies have shown that esophageal cancer is associated with fumonisin toxicity. However, the molecular mechanism of FB1-induced esophageal cancer is unclear. In this research, the molecular mechanism of FB1-induced cell carcinogenesis in human esophageal epithelial cells line (HEEC) was explored. We found that FB1 (0.3125–5 μM) could promote cell proliferation, and the same phenomenon was found in a 3D cell model. FB1 could also accelerate cell migration. The expression levels of DNA damage markers were significantly increased after FB1 exposure. Meanwhile, the expression levels of cell cycle-regulated proteins and cancer-related genes were abnormal. Furthermore, FB1 significantly upregulated the histone deacetylase (HDAC) expression and activated the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) signalling pathway. The HDAC inhibitor trichostatin A (TSA) could repressed FB1-promoted cell proliferation and abnormal phenomenon induced by FB1. Moreover, myriocin (ISP-1) could relieve FB1-enhanced HDAC expression and cell proliferation, which implied that ISP-1 may be used to block the fumonisin toxicity in the future. Our findings suggested that the HDAC/PI3K/Akt signalling pathway is a novel mechanism for FB1-induced cell carcinogenesis in HEEC and provided new ideas for the prevention and control of fumonisin toxicity, subsequently avoiding adverse effects on the ecosystem and human health.

Micro-Current Stimulation Has Potential Effects of Hair Growth-Promotion on Human Hair Follicle-Derived Papilla Cells and Animal Model.

Recently, a variety of safe and effective non-pharmacological methods have been introduced as new treatments of alopecia. Micro-current electrical stimulation (MCS) is one of them. It is generally known to facilitate cell proliferation and differentiation and promote cell migration and ATP synthesis. This study aimed to investigate the hair growth-promoting effect of MCS on human hair follicle-derived papilla cells (HFDPC) and a telogenic mice model. We examined changes in cell proliferation, migration, and cell cycle progression with MCS-applied HFDPC. The changes of expression of the cell cycle regulatory proteins, molecules related to the PI3K/AKT/mTOR/Fox01 pathway and Wnt/β-catenin pathway were also examined by immunoblotting. Subsequently, we evaluated the various growth factors in developing hair follicles by RT-PCR in MCS-applied (MCS) mice model. From the results, the MCS-applied groups with specific levels showed effects on HFDPC proliferation and migration and promoted cell cycle progression and the expression of cell cycle-related proteins. Moreover, these levels significantly activated the Wnt/β-catenin pathway and PI3K/AKT/mTOR/Fox01 pathway. Various growth factors in developing hair follicles, including Wnts, FGFs, IGF-1, and VEGF-B except for VEGF-A, significantly increased in MCS-applied mice. Our results may confirm that MCS has hair growth-promoting effect on HFDPC as well as telogenic mice model, suggesting a potential treatment strategy for alopecia.