Insulin-like growth factor-I prevents hypoxia-inducible factor-1 alpha-dependent G1/S arrest by activating cyclin E/cyclin-dependent kinase2 via the phoshatidylinositol-3 kinase/AKT/forkhead box O1/Cdkn1b pathway in porcine granulosa cells†.

As the follicle develops, the thickening of the granulosa compartment leads to progressively deficient supply of oxygen in granulosa cells (GCs) due to the growing distances from the follicular vessels. These conditions are believed to cause hypoxia in GCs during folliculogenesis. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo cell cycle arrest. However, it remains unclear whether hypoxia exerts any impact on cell cycle progression of GCs. On the other hand, although the GCs may live in a hypoxic environment, their mitotic capability appears to be unaffected in growing follicles. It thus raises the question whether there are certain intraovarian factors that might overcome the inhibitory effects of hypoxia. The present study provides the first evidence suggesting that cobalt chloride (CoCl2)-mimicked hypoxia prevented G1-to-S cell cycle progression in porcine GCs. In addition, we demonstrated that the inhibitory effects of CoCl2 on GCs cell cycle are mediated through hypoxia-inducible factor-1 alpha/FOXO1/Cdkn1b pathway. Moreover, we identified insulin-like growth factor-I (IGF-I) as an intrafollicular factor required for cell cycle recovery by binding to IGF-I receptor in GCs suffering CoCl2 stimulation. Further investigations confirmed a role of IGF-I in preserving G1/S progression of CoCl2-treated GCs via activating the cyclin E/cyclin-dependent kinase2 complex through the phoshatidylinositol-3 kinase/protein kinase B (AKT)/FOXO1/Cdkn1b axis. Although the present findings were based on a hypoxia mimicking model by using CoCl2, our study might shed new light on the regulatory mechanism of GCs cell cycle upon hypoxic stimulation.

NK007 helps in mitigating paclitaxel resistance through p38MAPK activation and HK2 degradation in ovarian cancer.

Ovarian cancer resistance to available medicines is a huge challenge in dire need of a solution, which makes its recurrence and mortality rate further exacerbated. A promising approach to overcome chemoresistance is drug screening from natural products. Here, we report that NK007, a (±)-tylophorine malate isolated from the Asclepiadaceae family, selectively inhibited the proliferation of A2780 and A2780 (Taxol) cells and migration of paclitaxel-sensitive and -resistant ovarian cancer cells. Interestingly, the decline of cell viability, including cell multiplication, clonality, and migration capacity was independent on cell apoptosis. At the molecular level, NK007 considerably induced G1/S arrest and upregulated the expression of phospho-p38 mitogen-activated protein kinase (p-p38MAPK). In addition, hexokinase 2 (HK2) protein degradation was considerably elevated in the presence of NK007, which resulted in the reduction of oxygen consumption rate and extracellular acidification rate. Altogether, our results indicate that NK007, an analog of tylophorine, can overcome paclitaxel (PTX) resistance through p38MAPK activation and HK2 degradation. As an effective, alternative antiresistance agent, NK007 exhibits a promising potential to treat PTX-resistant ovarian cancer.

Epithelial Cell Cycle Behaviour in the Injured Kidney.

Acute kidney injury (AKI), commonly caused by ischemia-reperfusion injury, has far-reaching health consequences. Despite the significant regenerative capacity of proximal tubular epithelium cells (PTCs), repair frequently fails, leading to the development of chronic kidney disease (CKD). In the last decade, it has been repeatedly demonstrated that dysregulation of the cell cycle can cause injured kidneys to progress to CKD. More precisely, severe AKI causes PTCs to arrest in the G1/S or G2/M phase of the cell cycle, leading to maladaptive repair and a fibrotic outcome. The mechanisms causing these arrests are far from known. The arrest might, at least partially, be attributed to DNA damage since activation of the DNA-damage response pathway leads to cell cycle arrest. Alternatively, cytokine signalling via nuclear factor kappa beta (NF-κß) and p38-mitogen-activated protein kinase (p38-MAPK) pathways, and reactive oxygen species (ROS) can play a role independent of DNA damage. In addition, only a handful of cell cycle regulators (e.g., p53, p21) have been thoroughly studied during renal repair. Still, why and how PTCs decide to arrest their cell cycle and how this arrest can efficiently be overcome remain open and challenging questions. In this review we will discuss the evidence for cell cycle involvement during AKI and development of CKD together with putative therapeutic approaches.

BIRC6 promotes hepatocellular carcinogenesis: interaction of BIRC6 with p53 facilitating p53 degradation.

The genes that encode inhibitor of apoptosis proteins (IAPs) are frequently overexpressed in human cancers. However, the expression pattern and clinical significance of BIRC6, a member of IAPs, in hepatocellular carcinoma (HCC) remains unclear. Here we investigated the role of BIRC6 in hepatocellular carcinogenesis. We used immunoblot and immunochemical analyses to determine the levels of BIRC6 in 7 hepatoma cell lines and 160 HCC specimens. We evaluated the proognostic value of BIRC6 expression and its association with clinical parameters. A lentivirus-mediated silencing method was used to knockdown BIRC6, and the biological consequences of BIRC6 silencing in three hepatoma cell lines were investigated in vitro and in vivo. We found that BIRC6 overexpression was significantly correlated with serum ALT level and HCC vascular invasion. Patients with positive BIRC6 expression in tumor tissue had a poor survival and a high rate of recurrence. BIRC6 knockdown remarkably suppressed cell proliferation, caused G1/S arrest and sensitized hepatoma cells to sorafenib-induced apoptosis in hepatoma cells, which was partly reversed by RNA interference targeting p53. The mechanistic study revealed that BIRC6 interacted with p53 and facilitated its degradation. The in vivo study showed that BIRC6 knockdown inhibited xenograft tumor growth and increased the sensitivity of tumor cells to sorafenib in nude mice. Taken together, these findings demonstrate that BIRC6 overexpression in HCC specimens is indicative of poor prognosis and that its interaction with p53 facilitates the degradation of p53, leading to carcinogenesis and an anti-apoptotic status.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants.

Alternol induces an S-phase arrest of melanoma B16F0 cells.

Alternol is a novel compound purified from the fermentation products of a microorganism in the yew tree bark. This study looks at the effects of alternol on the proliferation and cell cycle distribution of mouse melanoma cells. The inhibition of cell proliferation and changes in cell cycle distribution were analysed by sulforhodamine B and flow cytometry assays, respectively. mRNA expression of cyclin A, cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) and CDK inhibitor1A (p21) were measured by real-time reverse transcription PCR (RT-PCR). The protein levels of cyclin A, CDK2 and PCNA were analysed by Western blot analysis. p21 was measured by ELISA. Alternol treatment caused a significant decrease in the proliferation rate of B16F0 and B16F10 cells, which were significantly arrested in S phase, but this treatment had less effect on normal human embryonic kidney 293T cells. The mechanism by which alternol inhibits B16F0 proliferation in vitro may be associated with the inhibition of CDK2 and PCNA, and the activation of p21.

Homologous Recombination is Activated at Early Time Points Following Exposure to Cobalt Chloride Induced Hypoxic Conditions in Saccharomyces cerevisiae.

DNA repair functions are essential for the maintenance of genetic integrity and are regulated in response to both environmental and chemical stressors in mammalian and yeast cells in culture. The inhibitory effect of limited O2 availability on DNA repair functions in general and on homologous recombination (HR) in particular, correlates with increased chromosomal abnormalities in hypoxic cancer cells. Given the above, we have investigated the effects of CoCl2,--a hypoxia mimetic agent on HR and genetic aberrations in Saccharomyces cerevisiae. Our studies demonstrate that both acute and chronic exposure to CoCl2 activated HR and increased genetic aberrations in S. cerevisiae D7 cells. At early time points following addition of CoCl2 to the growth media, cells were briefly arrested in the G1-S boundary concomitant with a transient increase in Rad52-GFP foci formation and induction of low levels of DNA damage. The mode of action of CoCl2 is thus similar to that of DNA synthesis inhibitors, the later are known to induce HR and cause G1-S arrest. We propose that the activation of HR in the presence of the hypoxia mimetic agent may be attributed to the replication stress and/or DNA damage induced by the stressor.

RNA-binding protein MEX3A controls G1/S transition via regulating the RB/E2F pathway in clear cell renal cell carcinoma.

MEX3A is an RNA-binding protein that mediates mRNA decay through binding to 3' untranslated regions. However, its role and mechanism in clear cell renal cell carcinoma remain unknown. In this study, we found that MEX3A expression was transcriptionally activated by ETS1 and upregulated in clear cell renal cell carcinoma. Silencing MEX3A markedly reduced clear cell renal cell carcinoma cell proliferation in vitro and in vivo. Inhibiting MEX3A induced G1/S cell-cycle arrest. Gene set enrichment analysis revealed that E2F targets are the central downstream pathways of MEX3A. To identify MEX3A targets, systematic screening using enhanced cross-linking and immunoprecipitation sequencing, and RNA-immunoprecipitation sequencing assays were performed. A network of 4,000 genes was identified as potential targets of MEX3A. Gene ontology analysis of upregulated genes bound by MEX3A indicated that negative regulation of the cell proliferation pathway was highly enriched. Further assays indicated that MEX3A bound to the CDKN2B 3' untranslated region, promoting its mRNA degradation. This leads to decreased levels of CDKN2B and an uncontrolled cell cycle in clear cell renal cell carcinoma, which was confirmed by rescue experiments. Our findings revealed that MEX3A acts as a post-transcriptional regulator of abnormal cell-cycle progression in clear cell renal cell carcinoma.

AG-1024 Sensitizes Sorafenib-Resistant Hepatocellular Carcinoma Cells to Sorafenib via Enhancing G1/S Arrest.

PURPOSE: The frequency in resistance to sorafenib accounts for the grim prognosis of advanced hepatocellular carcinoma (HCC). In the present study, we explore the anti-cancer efficacy of co-administration of sub-toxic AG-1024 with sorafenib in HCC cells to enhance the sensitivity of these cells to sorafenib. MATERIALS AND METHODS: Two acquired sorafenib-resistant HCC cells, SNU-sora-5 and SK-sora-5, were established and verified. The MTT assay, colony formation assay, cell morphology detection and flow cytometric analysis were then used to determine the anti-tumor effects of the co-administration of sub-toxic AG-1024 and sorafenib. Finally, the potential molecular mechanism was preliminarily examined. RESULTS: Compared to parental cell lines, the acquired sorafenib-resistant cell lines, SNU-sora-5 and SK-sora-5, were more resistant to sorafenib. Sub-toxic AG-1024 markedly enhanced sorafenib-mediated cell inhibition in acquired sorafenib-resistant HCC strains, with a reversal index (RI) of 4.64 in SNU-sora-5 and 4.58 in SK-sora-5 cell lines. Moreover, co-administration of sub-toxic AG-1024 and sorafenib exerted dramatic cytotoxicity compared with sorafenib alone in the intrinsic sorafenib-resistant HCC-LM3 cells. In contrast to high-dose sorafenib, sub-toxic AG-1024 combined with sorafenib had less impact on apoptosis while significantly enhancing G1/S arrest via activation of the mTOR/p21 signaling pathway. The more, pharmacological inhibition of mTOR activity by inhibitor Palomid 529 significantly antagonized the synergistic anti-cancer effects of AG-1024 and sorafenib in HCC cells. CONCLUSION: The current findings indicate that sub-toxic AG-1024 may be a promising therapeutic agent in enhancing the sensitivity in HCC cells to sorafenib, bringing hope to HCC patients refractory to sorafenib treatment.

LOH12CR1 is a Novel Tumor Suppressor Inhibiting Tumor Growth Through Deregulation of G1/S Checkpoint in Human Colorectal Carcinoma.

BACKGROUND: Loss of heterozygosity (LOH) of 12p12-13 has been frequently found in various types of cancer. LOH12CR1 is one of the seven critical genes located within the 12p12-13 region. The protein encoded by LOH12CR1 is involved in the function of lysosomes and its other functions are still unclear. OBJECTIVE: The aim of this study is to investigate the potential roles of LOH12CR1 in the development of colorectal cancer. METHODS: A total of 174 colorectal cancer tissues were used to examine the protein level of LOH12CR1 by immunohistochemistry staining. The correlation between LOH12CR1 expression and the patient prognosis was further investigated through retrospective study. The tumor suppression capacity was examined by knockdown or overexpression of LOH12CR1 in four colorectal cancer cell lines and one normal cell line. RESULTS: Significant decrease of LOH12CR1 protein was observed in colorectal cancerous tissues (P<0.001). Knockdown of LOH12CR1 promoted colorectal cancer cell proliferation, colony formation, and accelerated G1/S cell cycle transition through downregulation of p16INK4a and p21WAF1/CIP1, while ectopic expression of LOH12CR1 displayed the opposite effects. The protein level of LOH12CR1 was well correlated with the expression of p16INK4a and p21WAF1/CIP1. Most importantly, the protein level of LOH12CR1 negatively correlated with clinical prognosis of colorectal carcinomas. CONCLUSION: The present results suggest that LOH12CR1 might function as a tumor suppressor. Thus, loss of function of LOH12CR1 might be a potential driver in the development of colorectal carcinoma. Detection of LOH12CR1 could be used as a method for diagnosis and therapeutic assessment of patients with colorectal cancer.

Ethanol extract of Ilex hainanensis Merr. exhibits anti-melanoma activity by induction of G1/S cell-cycle arrest and apoptosis.

OBJECTIVE: To evaluate anti-melanoma effect of ethanol extract of Ilex hainanensis Merr. (IME) and elucidate its underlying mechanism. METHODS: Thirty-six tumor-bearing mice were randomized into 6 groups (n=6) as follows: model group, IME 25, 50, 100, and 200 mg/kg groups and dacarbazine (DTIC) 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25, 50, 100 or 200 mg/kg per day, respectively. The mice in the DTIC group were intraperitoneally injected with DTIC 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The cell viability was evaluated by 3-(4,5-dime-thylthylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene and protein expressions of nuclear factor κB-p65 (NF-κB-p65), Bcl-2, B-cell lymphomaextra large (Bcl-xL) and Bax were detected by quantitative real-time polymerase chain reaction and Western blot analyses. Caspases-3, -8, and -9 activities were detected using the colorimetric method. In addition, a B16-F10 melanoma xenograft mouse model was used to evaluate the anti-cancer activity of IME in vivo. Furthermore, a survival experiment of tumor-bearing mice was also performed to evaluate the possible toxicity of IME. RESULTS: IME significantly inhibited the proliferation of B16-F10 cells (P<0.01). Flow cytometric analysis showed that IME induced G1/S cell cycle arrest and apoptosis (both P<0.01). IME inhibited activation of NF-κB, decreased the gene and protein expressions of Bcl-2, Bcl-xL, and increased the gene and protein expressions of Bax (all P<0.01). In addition, IME induced the activation of Caspases-3, -8, and -9 in B16-F10 cells. The study in vivo showed that IME significantly reduced tumor volume (P<0.01), and the inhibitory rate came up to 68.62%. IME also induced large areas of necrosis and intra-tumoral apoptosis that correlated with a reduction in tumor volume. Survival experiment showed that treatment with IME for 14 days significantly prolonged survival time and 20% of mice in the IME 200 mg/kg group were still alive until the 50th day. Notably, IME showed no apparent side-effects during the treatment period. CONCLUSION: IME exhibited significant anti-melanoma activity in vitro and in vivo, suggesting that IME might be a promising effective candidate with lower toxic for malignant melanoma therapy.

T cell proliferation and adaptive immune responses are critically regulated by protein phosphatase 4.

The clonal expansion of activated T cells is pivotal for the induction of protective immunity. Protein phosphatase 4 (PP4) is a ubiquitously expressed serine/threonine phosphatase with reported functions in thymocyte development and DNA damage responses. However, the role of PP4 in T cell immunity has not been thoroughly investigated. In this report, our data showed that T cell-specific ablation of PP4 resulted in defective adaptive immunity, impaired T cell homeostatic expansion, and inefficient T cell proliferation. This hypo-proliferation was associated with a partial G1-S cell cycle arrest, enhanced transcriptions of CDK inhibitors and elevated activation of AMPK. In addition, resveratrol, a known AMPK activator, induced similar G1-S arrests, while lentivirally-transduced WT or constitutively-active AMPKα1 retarded the proliferation of WT T cells. Further investigations showed that PP4 co-immunoprecipitated with AMPKα1, and the over-expression of PP4 inhibited AMPK phosphorylation, thereby implicating PP4 for the negative regulation of AMPK. In summary, our results indicate that PP4 is an essential modulator for T cell proliferation and immune responses; they further suggest a potential link between PP4 functions, AMPK activation and G1-S arrest in activated T cells.

Selective modulation of MAPKs contribute to the anti-proliferative and anti-inflammatory activities of 1,7-dihydroxy-3,4-dimethoxyxanthone in rheumatoid arthritis-derived fibroblast-like synoviocyte MH7A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: 1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is an antirheumatic agent isolated from traditional Chinese medicine Securidaca inappendiculata Hassk. This study was designed to investigate its anti-proliferative and anti-inflammatory activities on rheumatoid arthritis derived fibroblast-like synoviocyte cell line MH7A, and explore the underlying mechanism of action. METHODS: The anti-proliferative activity of XAN on MH7A cells was assessed by an MTT method. Its pro-apoptotic and cell cycle arrest activities were analyzed by flow cytometry. W-B method was employed to investigate hallmark kinases involved in the course. Pro-inflammatory cytokines in culture supernatant of MH7A cells were determined by an ELISA method. RESULTS: The results showed XAN efficiently suppressed the proliferation and secretion of IL-1ß and IL-6 of MH7A cells in a concentration-dependent manner. Co-treatment with MAPKs inhibitors U0126, SB202190 and SP600125 indicated JNK and p38 pathways were involved in the course. Up-regulation of p-p38, p-ERK, bax and p21, and down-regulation of p-JNK, cyclin D1 and bcl-2 were observed upon the treatment with XAN. SB202190 partly reversed the modulatory effects. The results suggested XAN inhibited the proliferation of MH7A cells mainly via cell cycle arrest at G1/S phase, and the activity was due to the up-regulation of p-p38, which led to the modulation of p21 and cyclin D1. The down-regulation of p-JNK by XAN suppressed the secretion of pro-inflammatory cytokines, which was beneficial to the anti-proliferative activity of MH7A cells. CONCLUSION: XAN selectively modulated MAPKs signaling, and exerted the subsequent anti-proliferative and anti-inflammatory activities on MH7A cells.

Tangeretin, a citrus pentamethoxyflavone, exerts cytostatic effect via p53/p21 up-regulation and suppresses metastasis in 7,12-dimethylbenz(α)anthracene-induced rat mammary carcinoma.

Breast cancer is the most commonly diagnosed cancer among women worldwide, which is characterized by unregulated cell growth and metastasis. Many bioactive compounds of plant origin such as tangeretin have been shown to possess potent antioxidant and anticancerous properties. In the present study we have investigated the chemotherapeutic effect of tangeretin against 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis and studied its underlying mechanism of action. Breast cancer was induced by "air pouch technique" with a single dose of 25mg/kg of DMBA. Tangeretin (50 mg/kg) was administered orally for four weeks. Remarkably, tangeretin treatment controlled the growth of cancer cells which was clearly evidenced by morphological and histological analysis. Also, serum levels of estradiol, progesterone and prolactin; lipid bound sialic acid and total sialic acid and the tissue levels of nitric oxide and protein carbonyls of cancer induced animals were decreased upon tangeretin treatment. Staining of breast tissues for nucleolar organizer regions, mast cells, glycoproteins, lipids and collagen showed that tangeretin treatment to breast cancer induced rats significantly reduced tumorigenesis. Oral tangeretin treatment also effectively reduced the tumor cell proliferation markers such as PCNA, COX-2 and Ki-67. Further, tangeretin treatment arrested the cancer cell division at the G1/S phase via p53/p21 up-regulation and inhibited metastasis by suppressing matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor. Taken together, the data provides new evidence on the mechanism of action of tangeretin in breast cancer and hence extends the hypothesis supporting its potential use in chemotherapy.

Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells.

In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0mM) for 12, 24 or 36h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5-4.0mM) and then decreased (8.0mM). Down-regulation of p53 by RNA interference increased 4.0mM of DES-induced apoptosis but did not affect 2.0mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure.