Curcumin Induces G2/M Cell Cycle Arrest In A P53-dependent Manner And Up-regulation ING4 Expression In Human Glioma

INTRODUCTION: Gliomas are the most common primary tumours of the central nervous system (CNS), which are histopathologically classified into gradeI, grade II (low grade), grade III (anaplastic) and grade IV (astrocytic phenotype or glioblastoma multiforme (GBM)) with increasing malignancy. Beginning in the 1980s chemotherapy has been incorporated into the treatment protocol of intractable glioma, and remains part of the treatment triad that includes microneurosurgery and radiation therapy. Unfortunately, for many intracranial neoplasms, effect of chemotherapy has failed to improve patient outcome significantly due to tumor heterogeneity and drug resistance to the traditional chemotherapy. The difficult clinical situation has fostered significant interest in defining novel therapeutic modalities for this heterogeneous group of neoplasms. During the initiation, progress and promotion of glioma, complicated gene mutations compose the intrinsic elements, such as activation of oncogenes or inactive functional consequences of mutations in tumor suppressor genes. Cells become cancerous when they acquire genetic alterations that allow them to evade the normal proliferation tightly regulated by multiple signals. ING (inhibitor of growth) gene family including the members ING1, ING2, ING3, ING4 and ING5, is characterized by a highly conserved C-terminal plant homeodomain (PHD)-like zinc-finger domain and implicated in a variety of processes including oncogenesis, apoptosis, DNA repair, and cell cycle control. The best-studied ING family members reside in the nucleus where they positively or negatively regulate gene expression through interactions between their N terminus and chromatin-remodeling complexes containing histone acetyl-transferases, histone deacetyltransferases, or factor acetyltransferases. ING1 and ING2 can negatively regulate cell proliferation in a p53-dependent manner through induction of G1 phase arrest of cell cycle and apoptosis, where ING1b can physically associate with p53 and promote apoptosis. ING2 increases the acetylation of p53 at Lys-382 and strongly enhances the transcriptional-transactivation activity of p53 although there is no physical interaction between both the proteins. ING3 can activate p53-transactivated promoters, including promoters of p21 and Bax; and meanwhile, the overexpression of ING3 induces a decreased population of cells in S phase and apoptosis. Transient overpression of ING5 in RKO cells can induce decrease in S phase of cell cycle and apoptosis in a p53-dependent manner with the upregulation of p21.ING4 (GenBank Accession No. AF110645 or NM₀16162) was initially identified in a screen for novel tumor suppressors, whose overexpression suppressed loss of contact inhibition but did not inhibit cell proliferation and not directly affect the cell cycle. It's a novel member of ING family, characterized as a candidate tumor suppressor gene that frequently mutated in human cancer. ING4 can increase the acetylation of p53 via interaction with p300, negatively regulate the cell growth with significant G2/M arrest of cell cycle in HepG2 cells, and enhance the sensitivity of some tumor cells to DNA-damage drugs. Recent study has shown that ING4 is involved in regulating brain tumour growth and angiogenesis. Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. ING4 physically interacts with p65 (RelA) subunit of nuclear factor NF-kB, and that ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-kB-responsive genes. The inhibition of ING4 expression can significantly up-regulate the expression of COX-2, indicating the close relationship between ING4 and COX-2. ING4 has an important role in brain tumour pathogenesis.Curcumin is a diferuloylmethane derived from the plant Curcuma longa. Accumulating evidences from the labs and clinical trials have show that it is a promising chemopreventive agent belonging to two categories: (i) blocking agents, which inhibit the initiation step by preventing carcinogen activation and (ii) suppressing agents, which inhibit malignant cell proliferation during promotion and progression steps of carcinogenesis. And available experimental evidence also indicate it as a as a cancer therapeutic agent. Timiras PS. etc. firstly reported that curcumin capably suppressed the proliferation and growth of glioma cells in vitro in a time-dose- dependent manner. Kim HS. etc. found that curcumin strongly repressed MMP-1, -3 and -9 gene expression via the inhibition of the PKC to MAP kinase signaling pathways and the repression of the DNA-binding and transcription activities of AP-1 in human astroglioma cells, and also significantly repressed the in vitro invasion of glioma cells. These studies strongly suggest that curcumin might provide a novel therapeutic strategy for treating glioma. In addition, curcumin is believed to be pharmacologically safe because it is a naturally occurring substance that is used in both the diet as well as in traditional oriental medicine to treat various diseases in East-Asian countries as well as in India. However the precise molecular mechanisms underlying the anti-tumor effects of curcumin on glioma have remained unresolved.AIM: To detect the expression of ING4 in normal brain tissues and glioma tissues of different grades in malignancy, and discuss the relationship between ING4 and tumorigenesis, malignancy and prognosis of glioma. To observe the inhibitory effects of curcumin on cell growth and proliferation of human glioma cells in vitro. To observe the effects of low-dose curcumin on cell cycle distribution of glioma cells, and reveal the potential molecular mechanisms. To observe the effects of high-dose curcumin on induction of apoptosis in glioma cells, and reveal the apoptotic signal pathway.METHODS: Part I (1) Immunohistochemistry staining was used to examine the expression of ING4 in normal brain tissues and glioma tissues. (2) Western blot was used to examine the protein expression of ING4 in normal brain tissues and glioma tissues. (3) Immunocyto-fluorescence staining was used to examine the expression and subcellular distribution of ING4 in the human glioma cell lines U251 and SGH44. Part II (1) MTT assay was performed to examine the dose effects of curcumin on cell growth and proliferation of human glioma cells in vitro. (2) RT-PCR and Western blot analysis were used to examine the mRNA and protein expressions of ING4 in human glioma cell lines. (3) FACS analyzed the changes in cell cycle distributions of glioma cells after the treatments of low-dose curcumin. (4) DNA ladder gel electrophoresis was used to assess apoptosis induced by low-dose of curcumin in glioma cells. (5) Western blot was performed to examine the changes in p53, p21^waf1/cip1, CyclinB1, Cdc2 and ING4 protein expressions after the treatment of low-dose curcumin. (6) Immunocyto- fluorescence staining was performed to detect the expression of ING4 in glioma cells after the treatment of low-dose curcumin. Part III (1) MTT assay was performed to examine the dose-time-effects of curcumin on cell growth and proliferation of human glioma cells in vitro. (2) FACS analyzed the changes in cell cycle distributions of glioma cells after the treatments of high-dose curcumin. (3) DNA ladder gel electrophoresis and FACS were used to assess apoptosis induced by high-dose of curcumin in glioma cells. (4) Western blot analyzed the protein expressions of p53, p21^waf1/cip1 , Bcl-2, Bax, Bak, cytochrome C, and p27kip1 in SC236- and MK886-induced apoptosis. (5) Western blot was performed to examine the changes in p53, p21^waf1/cip1, ING4, Bcl-2 family members, caspapse-3 and PARP protein expressions after the induction of apoptosis by high-dose curcumin. (6) Immunoassay was performed to detect the release of cytochrome C from the mitochondria to cytoplasm in glioma cells after the treatment of low-dose curcumin.RESULTS: Part I (1) Expression of ING4 was strong or moderate positively expressed in the normal brain tissues, and significantly reduced in gliomas as compared with normal human brain tissue. (2) In astrocytoma tissues ING4 was negatively or weak positively expressed, and there were not significant differences in the ING4 positive expression rate between grade I and grade II glioma. However in grade III and grade IV gliomas, the expression of ING4 was obviously reduced, that the extent of reduction in the expression intensity and range was correlated with the progression from lower to higher grades of tumors. (3) Western blot showed that ING4 was weakly expressed in gliomas in contrast to the strong positively expression in normal human brain tissue. (4) In human glioma cell lines U251 and SGH44, the expression of ING4 was almost undetectable. Part II (1) ING4 mRNA was hardly detectable inU251 cells, but weakly expressed in SGH44 cells. And ING4 protein expression in U251 cells was undetectable. (2) Curcumin effectively inhibited cell growth of human glioma cells U251 in vitro in a dose-dependent manner. (3) Low doses of curcumin affected the cell cycle distributions, increasing the number of cells in S phase and G2/M phase. But no significant apoptotic peak was induced. (4) DNA electrophoresis did not show obvious DNA ladder formation after the treatment of low doses curcumin in U251 cells. (5) Western blot showed that the expression of p53 and p21waf1/cip1 were up-regulated,and the expression of Cyclin B1 and Cdc2 were down-regulated, and the expression of ING4 was increased by low-dose curcumin in U251 cells. (6) Immunocyto-fluorescence staining detected expression of ING4 in nucleolus of U251 cells after the treatment of low-dose curcumin. Part III (1) MTT showed that curcumin inhibited cell growth of human glioma cells U251 in vitro in a dose-time-dependent manner. (2) ING4 mRNA was hardly detectable inU251 cells, but weakly expressed in SGH44 cells. And ING4 protein expression in U251 cells was undetectable. (2) FACS showed that high-dose curcumin induced cell cycle arrest in G0/G1 phase and significantly induced apoptosis in U251 cells. (3) DNA electrophoresis showed obvious DNA ladder formation after the treatment of high doses curcumin in U251 cells. And FACS showed after the treatment of high-dose curcumin, 41.3% of U251 cells were induced apoptosis. (4) Western blot showed that the expression of p53 and p21waf1/cip1 were up-regulated,and the expression of Bcl-2 was significantly down-regulated, but the expression of Bax and Bak were not changed, which lead to the ratio of Bcl-2: Bax was decreased. And the expression of ING4 was increased by high-dose curcumin in U251 cells. ING4 protein expression was undetectable with or without the treatment of curcumin. (5) After the treatment of high-dose curcumin, the cytochrome C release into cytoplasm were up-regulated, and caspase-3 was activated with the induced appearance of the p17 subunit, and the p85 subunit cleavage product of PARP (p116), which was cleaved when caspase-3 was activated, was generated.CONCLUSION: (1) ING4 was strong positively expressed in normal brain tissues as compared to weakly or negatively expressed in glioma tissues, and the extent of reduction in the ING4 expression intensity and range was correlated with the progression from lower to higher grades of tumors. (2) Curcumin inhibited cell growth and proliferation of glioma cell in vitro in a dose-time-dependent manner. (3) The low doses (< IC50) of curcumin induced cell cycle arrest in S phase and G2/M phase without apoptosis induced, however the high doses (> IC50) of curcumin induced cell cycle arrest in G0/G1 phase and significant apoptosis. (4) Low and high doses of curcumin induced different cellular responses via respective signal pathway. P53 was the potential common factor playing key roles in the different cell processes, but ING4 possibly only was involved in cell cycle arrest. (5) Low dose of curcumin significantly up-regulated the expressions of p53 and p21^waf1/cip1, down-regulated CyclinB1 and Cdc2 expression, and induced ING4 expression, which indicated that curcumin possibly suppressed CyclinB1 and Cdc2 expression in a p53-dependent manner and leaded to cell cycle arrest at G2/M phase. And ING4 potentially function on the regulation of cell cycle via the interaction with p53. (6) High-dose curcumin induced apoptosis through up-regulation of p53 and p21^waf1/cip1, down-regulation of Bcl-2 and the ratio of Bcl-2: Bax, increase in cytochrome C release and final activation of caspase-3. Further studies to clarify the functions and molecular mechanisms of curcumin in glioma cells will establish the useful base for the exploration of its clinical value and offer more potential target molecules as candidates in chemotherapy and chemoprevention of glioma.