Suppression of bcl-2 gene by RNA interference increases chemosensitivity to cisplatin in nasopharyngeal carcinoma cell line CNE1

To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE1     

Tetrandrine enhances radiosensitivity through the CDC25C/CDK1/cyclin B1 pathway in nasopharyngeal carcinoma cells

The increasing resistance of nasopharyngeal carcinoma to irradiation makes the exploration of effective radiosensitizers necessary. Tetrandrine is known to be an antitumor drug, but little is known regarding its radiosensitization effect on nasopharyngeal carcinoma. We investigated the effect of combined treatment of irradiation and maximum non-cytotoxic doses of tetrandrine on the nasopharyngeal carcinoma cell lines CNE1 and CNE2. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The radiosensitization of cells receiving the maximum non-cytotoxic doses of tetrandrine was assessed by evaluating cell proliferation and DNA damage repair using MTT, clonogenic, comet assays and detection of caspase-3 and phosphorylated histone H2AX (γ-H2AX). The cell cycle was assessed by flow cytometry, and protein expression was detected by western blot analysis. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were 1.5 μmol/L and 1.8 μmol/L, respectively. When cells were exposed to irradiation and the maximum non-cytotoxic doses of tetrandrine, the survival fraction was decreased. DNA damage and γ-H2AX levels markedly increased. Moreover, tetrandrine abrogated the G2/M phase arrest caused by irradiation. Combined treatment with the maximum non-cytotoxic dose of tetrandrine and irradiation caused suppression of the phosphorylation of CDK1 and CDC25C and increase in the expression of cyclin B1. The study in vivo also showed that the maximum non-cytotoxic dose of tetrandrine could reduce tumor growth in xenograft tumor model. Our results suggest that the maximum non-cytotoxic dose of tetrandrine can enhance the radiosensitivity of CNE1 and CNE2 cells and that the underlying mechanism could be associated with abrogation of radiation-induced G2/M arrest via activation of the CDC25C/CDK1/Cyclin B1 pathway.     

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.     

Geraniol inhibits prostate cancer growth by targeting cell cycle and apoptosis pathways

The progression of prostate cancer is associated with escape from cell cycle arrest and apoptosis under androgen-depleted conditions. Here, we found that geraniol, a naturally occurring monoterpene, induces cell cycle arrest and apoptosis in cultured cells and tumor grafted mice using PC-3 prostate cancer cells. Geraniol modulated the expression of various cell cycle regulators and Bcl-2 family proteins in PC-3 cells in vitro and in vivo. Furthermore, we showed that the combination of sub-optimal doses of geraniol and docetaxel noticeably suppresses prostate cancer growth in cultured cells and tumor xenograft mice. Therefore, our findings provide insight into unraveling the mechanisms underlying escape from cell cycle arrest and apoptosis and developing therapeutic strategies against prostate cancer.     

Garlic Compound, Diallyl Disulfide Induces Cell Cycle Arrest in Prostate Cancer Cell Line PC-3

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. Previous studies reported that aged garlic extract suppresses cancer growth. In the present study, diallyl disulfide [DADS], oil soluble organosulfur compound of garlic, was studied for its antiproliferative and induction of cell cycle arrest on prostate cancer cells in vitro. The suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed and confirmed by propidium iodide staining in flowcytometric analysis and western blotting analysis of major cell cycle regulator proteins. The results showed that DADS inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 25 μM and 40 μM concentrations, DADS induced cell cycle arrest at G2/M transition in PC-3 cells. Western blotting analysis of cyclin A, B₁ and cyclin dependent kinase 1 [CDK1] revealed that DADS inhibited the cell cycle by downregulating CDK1 expression. It is concluded that DADS, inhibits proliferation of prostate cancer cells through cell cycle arrest. Dose dependent effect of DADS on PC-3 cell line was observed in the present study.     

DNA repair pathways-targeted cyclovirobuxine inhibits castration-resistant prostate cancer growth by promoting cell apoptosis and cycle arrest

BackgroundCastration-resistant prostate cancer (CRPC) is a deadly malignancy without effective therapeutics. Cyclovirobuxine (CVB) can play an anticancer role by inhibiting mitochondrial function, regulating tumor cell apoptosis, dysregulating autophagy, and other mechanisms. This study aimed to examine the function and mechanism of CVB in CRPC to provide new insights into CRPC treatment.MethodsThe effect of CVB on PC3 and C4-2 cell viability was determined using a CCK8 assay. Core therapeutic targets of CVB in CRPC cells were identified using RNA sequencing, online database, and PPI network analyses. Western blotting, RT–qPCR and molecular docking were performed to evaluate the regulation of core targets by CVB. Utilizing GO and KEGG enrichment analyses, the probable anti-CRPC mechanism of CVB was investigated. Immunofluorescence, flow cytometry and colony formation assays were used to verify the potential phenotypic regulatory role of CVB in CRPC.ResultsCVB inhibited CRPC cell activity in a concentration-dependent manner. Mechanistically, it primarily regulated BRCA1-, POLD1-, BLM-, MSH2-, MSH6- and PCNA-mediated mismatch repair, homologous recombination repair, base excision repair, Fanconi anemia repair, and nucleotide excision repair pathways. Immunofluorescence, Western blot, flow cytometry and colony formation experiments showed that CVB induced DNA damage accumulation, cell apoptosis, and cell cycle arrest and inhibited CRPC cell proliferation.ConclusionCVB can induce DNA damage accumulation in CRPC cells by targeting DNA repair pathways and then induce cell apoptosis and cell cycle arrest, eventually leading to inhibition of the long-term proliferation of CRPC cells.     

三氧化二砷诱导CNE1凋亡及其对细胞周期的影响

目的 研究三氧化二砷对人鼻咽癌CNE1细胞凋亡及其细胞周期的影响。方法 应用形态学观察、原位末端标记法(TUNEL)、流式细胞术等方法对三氧化二砷诱导的鼻咽癌细胞CNE1进行检测和观察。结果 一定浓度三氧化二砷能诱导CNE1细胞凋亡,凋亡细胞具有典型的凋亡形态特征,TUNEL原位检测有典型凋亡细胞,流式细胞仪检测有凋亡峰,G2／M期比例升高,呈一定的剂量效应关系。结论 三氧化二砷能诱导人鼻咽癌CNE1细胞株凋亡及阻止细胞周期进展的作用。     

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells

Background: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.Material and methods: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.Results: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.Conclusion: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.     

蛇床子素通过促进胃癌细胞N87凋亡和细胞周期阻滞而抑制细胞增殖

蛇床子素是从伞形科植物蛇床中提取的一类具有生物活性的化合物。研究显示,蛇床子素对多种肿瘤细胞具有抑制作用,然而尚未有研究揭示其对胃癌N87细胞的抗肿瘤活性。本文研究了蛇床子素在体外和荷瘤小鼠体内对胃癌N87细胞的抗肿瘤效应,并进一步利用流式细胞术、TUNEL试验及Western印迹检测分析其对细胞周期及细胞凋亡的影响,以探索其作用机制。研究结果表明,蛇床子素有效地抑制了体外培养的N87细胞生长,并呈浓度依赖效应。本文还建立了N87的荷瘤小鼠模型。结果显示,无论是在低剂量(50 mg/kg)或高剂量(100 mg/kg)情况下,蛇床子素均显示了有效的肿瘤生长抑制效果。流式细胞术及Western印迹的结果表明,蛇床子素诱导N87细胞阻滞在G_2/M期。通过流式细胞术、TUNEL测试及Western印迹结果证明,蛇床子素通过激活胱天蛋白酶-3依赖的凋亡通路,最终导致了N87细胞凋亡的发生。综上所述,本研究显示,蛇床子素在胃癌N87细胞中通过促进细胞凋亡而发挥其抗肿瘤活性,这将为其应用于胃癌的临床治疗提供理论参考。     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

The 3-deoxysappanchalcone induces ROS-mediated apoptosis and cell cycle arrest via JNK/p38 MAPKs signaling pathway in human esophageal cancer cells

BackgroundThe 3-deoxysappanchalcone (3-DSC), a chemical separated from Caesalpinia sappan L, has been substantiated to display anti-inflammatory, anti-influenza, and anti-allergy activities according to previous studies. However, the underlying mechanisms of action on esophageal cancer remain unknown.PurposeThe present research aims to survey the action mechanisms of 3-DSC in esophageal squamous cell carcinoma (ESCC) cells in vitro.MethodsEvaluation of cytotoxicity was determined by MTT tetrazolium salt assay and soft agar assay. Cell cycle distribution, apoptosis induction, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), and multi-caspases activity were appreciated by Muse™ Cell Analyzer. The expressions of cell cycle- and apoptosis-related proteins were presented using Western blotting.Results3-DSC blocked cell growth and colony formation ability in a concentration-dependent manner and invoked apoptosis, G2/M cell cycle arrest, ROS production, MMP depolarization, and multi-caspase activity. Furthermore, Western blotting results demonstrated that 3-DSC upregulated the expression of phospho (p)-c-jun NH2-terminal kinases (JNK), p-p38, cell cycle regulators, pro-apoptotic proteins, and endoplasmic reticulum (ER) stress-related proteins whereas downregulated the levels of anti-apoptotic proteins and cell cycle promoters. The effects of 3-DSC on ROS induction were counteracted by pretreatment with N-acetyl-L-cysteine (NAC). Also, our results indicated that p38 (SB203580) and JNK (SP600125) inhibitor slightly inhibited 3-DSC-induced apoptosis. These results showed that 3-DSC-related G2/M phase cell cycle arrest and apoptosis by JNK/p38 MAPK signaling pathway in ESCC cells were mediated by ROS.ConclusionROS generation by 3-DSC in cancer cells could be an attractive strategy for apoptosis of cancer cells by inducing cell cycle arrest, ER stress, MMP loss, multi-caspase activity, and JNK/p38 MAPK pathway. Our findings suggest that 3-DSC is a promising novel therapeutic candidate for both prevention and treatment of esophageal cancer.     

Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 Has a Therapeutic Potential and Sensitizes Cisplatin in Nasopharyngeal Carcinoma

Phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells and thus has been considered as a promising drug target. To ascertain a therapeutical approach of nasopharyngeal carcinoma (NPC), we hypothesized NVP-BEZ235, a novel and potent imidazo[4,5-c] quinolone derivative, that dually inhibits both PI3K and mTOR kinases activities, had antitumor activity in NPC. Expectedly, we found that NVP-BEZ235 selectively inhibited proliferation of NPC cells rather than normal nasopharyngeal cells using MTT assay. In NPC cell lines, with the extended exposure, NVP-BEZ235 selectively inhibited proliferation of NPC cells harboring PIK3CA mutation, compared to cells with wild-type PIK3CA. Furthermore, exposure of NPC cells to NVP-BEZ235 resulted in G1 growth arrest by Propidium iodide uptake assay, reduction of cyclin D1and CDK4, and increased levels of P27 and P21 by Western blotting, but negligible apoptosis. Moreover, we found that cisplatin (CDDP) activated PI3K/AKT and mTORC1 pathways and NVP-BEZ235 alleviated the activation by CDDP through dually targeting PI3K and mTOR kinases. Also, NVP-BEZ235 combining with CDDP synergistically inhibited proliferation and induced apoptosis in NPC cells. In CNE2 and HONE1 nude mice xenograft models, orally NVP-BEZ235 efficiently attenuated tumor growth with no obvious toxicity. In combination with NVP-BEZ235 and CDDP, there was dramatic synergy in shrinking tumor volumes and inducing apoptosis through increasing Noxa, Bax and decreasing Mcl-1, Bcl-2. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential as a monotherapy or in combination with CDDP for NPC treatment.     

Insulin-like factor binding protein-3 promotes the G1 cell cycle arrest in several cancer cell lines

Insulin-like growth factor binding protein-3 (IGFBP-3) is a multi-functional protein known to induce apoptosis of various cancer cells in an insulin-like growth factor (IGF)-dependent and IGF-independent manner. In our previous study, we found that IGFBP-3 induced apoptosis through the activation of caspases in 786-O cells. In this study, we further examined that whether IGFBP-3 induced apoptosis through the induction of cell cycle arrest in 786-O, A549 and MCF-7 cells. Our results showed that overexpressed IGFBP-3 resulted in typical apoptotic ultrastructures in A549 cells under transmission electron microscope. The result of flow cytometry analysis indicated that IGFBP-3 arrested the cell cycle at G1-S phase in 786-O, A549 and MCF-7 cells. In A549 cells, quantitative real-time PCR and Western blot analysis showed a significant change in the expression of cell cycle-regulated proteins—a decrease in cyclin E1 expression, an increase in p21 expression. These results indicate a possible mechanism for G1 cell cycle arrest by IGFBP-3. Taken together, cyclin E1 and p21 may play important roles in the IGFBP-3-inducing G1 cell cycle arrest and apoptosis in several human cancer cells.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.     

The tumor supressor function of STGC3 and its reduced expression in nasopharyngeal carcinoma

STGC3 is a novel candidate tumor suppressor gene that was found to be associated with nasopharyngeal carcinoma (NPC) via the cDNA cloning and RACE processes. The biological function of the STGC3 protein and its expression level in nasopharyngeal carcinoma remain unknown. This study aimed to evaluate the STGC3 protein expression level in NPC and to investigate the inhibitory function of STGC3 as a candidate tumor suppressor gene. We assessed the expression of the STGC3 protein in NPC biopsies and normal control specimens via Western blot and immunohistochemical analysis. The expression of STGC3 as induced by doxycycline (Dox) via a tetracycline (Tet)-regulated system in human nasopharyngeal carcinoma cell line CNE2 was also established, and the effect of STGC3 restoration on the biological behavior of CNE2 was observed. A reduced level of STGC3 expression (0.978 ± 0.213 versus 0.324 ± 0.185, P < 0.05) was detected in NPC versus normal nasopharyngeal tissue by Western blot assay. Immunohistochemical assays for STGC3 detected positive staining in the nuclei and cytoplasm of epithelial cells, and the positive expression rate in NPC, 8 of 21 (38%), was lower than that in normal nasopharynx samples, 16 of 22 (72%). After STGC3 expression was restored, the growth capacity and clone formation potential of CNE2 cells in soft agar were significantly suppressed, and the cell percentage in G₀/G₁ phase increased, while the percentage of cells entering the S and G₂ phases decreased. This indicates that an abnormality in STGC3 expression is associated with nasopharyngeal carcinogenesis and that it may play an important role in controlling cell growth and regulating the cell cycle.     

Diabetes-induced abnormalities of mitochondrial function in rat brain cortex: the effect of n-3 fatty acid diet

High expression of connexins was found in a variety of cancers, but their role is still controversial. We investigated whether connexin43 (Cx43) contributed to bladder carcinogenesis through MAPK activation. In this study, we found that Cx43 expression was significantly increased in bladder cancer tissues and cell line. Overexpression of Cx43 in bladder cancer 5637 cells increased cell proliferation, promoted cell cycle progression, and inhibited apoptosis. Western blot showed that JNK and ERK pathways were dramatically activated in Cx43-overexpressed cells. Conversely, knockdown of Cx43 inhibited cell proliferation by increasing apoptosis and causing cell cycle arrest, concomitant with inhibition of JNK and ERK signaling. In addition, JNK and ERK pathways were also activated in bladder cancer tissues. In conclusion, abnormal high expression and cytoplasmic localization of Cx43 contributed to bladder cancer. Inhibition of Cx43 activity could be a potential therapeutic strategy for preventing the progression of bladder cancer.     

6-O-Angeloylenolin induced cell-cycle arrest and apoptosis in human nasopharyngeal cancer cells

High incidence of lymph node spread and distant metastasis make poor prognosis in human nasopharyngeal carcinoma (NPC). Therefore, better treatments for NPC are needed. This study investigated the anticancer activity of 6-O-angeloylenolin, a plant-derived sesquiterpene lactone, on human nasopharyngeal cancer (CNE) cells. 6-O-Angeloylenolin was found to significantly inhibit the proliferation of CNE cells. The rate of inhibition was comparable to that of cisplatin, a well known chemotherapeutic agent used to treat NPC. Further mechanistic studies revealed that 6-O-angeloylenolin caused cell-cycle arrest in the S and G2/M phases and, subsequently, the induction of apoptosis. Rapid repressions of cyclin D1, cyclin D3, p27, cdc25c and p-cdc25c (Ser216) were observed after 1-h treatment, followed by decreases in the expression of CDK4, cdc2 and p-cdc2 (Tyr15) after 12 h. Down-regulations of p-Rb (Ser780), p21^Waf1/Cip1, cyclin A, and cyclin E were also detected as later events. Two early events that marked the occurrence of apoptosis were phosphatidylserine exposure and mitochondria membrane potential depletion, which occurred after 12 h of treatment, while a sub-G1 peak was also detected after 36-h treatment. Apoptosis induction was further confirmed by other apoptotic features, including nuclear fragmentation, and PARP cleavage. Moreover, 6-O-angeloylenolin caused the release of cytochrome c and AIF to the cytosol by regulating the expression of the Bcl-2 family proteins. However, pretreatment of the general caspase inhibitor failed to attenuate the apoptosis induction effect, suggesting that apoptosis induction of 6-O-angeloylenolin was independent of caspase activation. While 6-O-angeloylenolin also triggered the activation of Akt, ERK and JNK, only the JNK inhibitor significantly decreased the extent of cell death and apoptosis in CNE cells. Taken together, these results suggest the potential applicability of 6-O-angeloylenolin as a candidate for NPC treatment.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.