Curcumin induced apoptosis is mediated through oxidative stress in mutated p53 and wild type p53 colon adenocarcinoma cell lines

Curcumin has anti‐oxidant, anti‐cancer and anti‐carcinogen property. Our laboratory had previously reported that, curcumin treatment induces reactive oxygen species (ROS) generation in HT‐29 cell line, an effect contradictory to its anti‐oxidant property. This study evaluates the role of p53 in curcumin mediated ROS generation and cell death. Curcumin induced ROS was determined by 2’,7’‐dichlorofluorescein and apoptosis by Hoechst33342/PI staining in HT‐29 and HCT‐116 cell lines. ROS generation occurs within 1 hour of 40 µM curcumin treatment and a reduction was observed by third hour in HCT‐116 insinuating p53 involvement. N‐acetyl cysteine (NAC) pre‐treatment effectively quenched ROS and inhibited membrane potential loss in HT‐29, but less effective in HCT‐116. Mitochondrial membrane potential loss is evident with 10 and 40 µM curcumin in HCT‐116 and at 40 µM curcumin in HT‐29. Total p53 protein level increase was observed by 24 hours in HCT‐116 upon NAC pre‐treatment. Our results indicate that curcumin induces ROS mediated cell death in colon adenocarcinoma cell lines and may be mediated via p53.     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.     

Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53

It has been shown that p53 induces cell apoptosis and the Bcl-2 family plays key roles in this process. However, the molecular mechanism of p53 apoptotic pathway is still unclear. Here, we show that overexpression of exogenous wild-type p53 induced apoptosis in lung cancer cells and high metastasis potential cells had a faster rate of apoptosis than low metastasis potential cells. The expression of pro-apoptotic gene BNIP3 was increased significantly both in Anip973 and 95D cell lines which have high metastasis ability, but not AGZY83-a or little increased in 95C cell lines which possess low metastasis ability. Overexpression of BNIP3 increases apoptotic rate induced by p53 in AGZY83-a cells. Blocking the expression of BNIP3 by siRNA in Anip973 cells decreased apoptotic rate mediated by p53. Taken together, these data suggest that high level expression of BNIP3 mediated rapid apoptosis that was triggered by p53 in lung cancer cells.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

miR-3133 inhibits gastrointestinal cancer progression through activation of Hippo and p53 signalling pathways via multi-targets

Background

Malignant cell growth and chemoresistance, the main obstacles in treating gastrointestinal cancer (GIC), rely on the Hippo and p53 signalling pathways. However, the upstream regulatory mechanisms of these pathways remain complex and poorly understood.

Methods

Immunohistochemistry (IHC), western blot and RT-qPCR were used to analyse the expression of RNF146, miR-3133 and key components of Hippo and p53 pathway. CCK-8, colony formation, drug sensitivity assays and murine xenograft models were used to investigate the effect of RNF146 and miR-3133 in GIC. Further exploration of the upstream regulatory mechanism was performed using bioinformatics analysis, dual-luciferase reporter gene, immunoprecipitation assays and bisulfite sequencing PCR (BSP).

Results

Clinical samples, in vitro and in vivo experiments demonstrated that RNF146 exerts oncogenic effects in GIC by regulating the Hippo pathway. Bioinformatics analysis identified a novel miRNA, miR-3133, as an upstream regulatory factor of RNF146. fluorescence in situ hybridization and RT-qPCR assays revealed that miR-3133 was less expressed in gastrointestinal tumour tissues and was associated with adverse pathological features. Functional assays and animal models showed that miR-3133 promoted the proliferation and chemotherapy sensitivity of GIC cells. miR-3133 affected YAP1 protein expression by targeting RNF146, AGK and CUL4A, thus activating the Hippo pathway. miR-3133 inhibited p53 protein degradation and extended p53's half-life by targeting USP15, SPIN1. BSP experiments confirmed that miR-3133 promoter methylation is an important reason for its low expression.

Conclusion

miR-3133 inhibits GIC progression by activating the Hippo and p53 signalling pathways via multi-targets, including RNF146, thereby providing prognostic factors and valuable potential therapeutic targets for GIC.     

The role of p53 in treatment responses of lung cancer

Resistance to radio- and chemotherapy is a major problem in treatment responses of lung cancer. In this disease, biological markers, that can be predictive of response to treatment for guiding clinical practice, still need to be validated. Radiotherapy and most chemotherapeutic agents directly target DNA and in response to such therapies, p53 functions as a coordinator of the DNA repair process, cell cycle arrest, and apoptosis. In fact, it participates in the main DNA repair systems operative in cells, including NHEJ, HRR, NER, BER, and MMR. Given the high p53 mutation frequency in lung cancer which likely impairs some of the p53-mediated functions, a role of p53 as a predictive marker for treatment responses has been suggested. In this review, we summarize the conflicting results coming from preclinical and clinical studies on the role of p53 as a predictive marker of responses to chemotherapy or radiotherapy in lung cancer.     

Serum withdrawal and etoposide induce apoptosis in human lung carcinoma cell line A549 via distinct pathways

The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposide-treated cells, but not in cells subjected to serum with-drawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypo-phosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid- induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.     

Small-molecule CB002 restores p53 pathway signaling and represses colorectal cancer cell growth

Much effort is currently focused on the p53 pathway. p53 is a key tumor suppressor, which is mutated or lost in many human cancers. Restoration of the p53 pathway holds the potential to induce selective cell death in tumor cells without harming normal cells that have intact p53 pathways. Most tumor cells express mutated p53 or suppress p53 by overexpression of MDM2. In this study, a compound referred to as CB002 with one closely related compound from the Chembridge library were evaluated for tumor cytotoxicity without affecting normal cells by restoration of the p53 pathway. A decrease of mutant p53 protein expression, restoration of inactivated p53, or some activation of p73 are candidate mechanisms this agent could cause tumor cell apoptosis and growth arrest. We further show that CB002 activates p53 pathway signaling in part via p73 in p53 mutant cancer cell lines. However, it is important to note that we have not established a role for p73 in the anti-tumor effect of CB002 or R1. CB002 causes tumor cell death with synergistic effects with traditional chemotherapeutics CPT-11 and 5-FU.     

Encorafenib enhances TRAIL-induced apoptosis of colorectal cancer cells dependent on p53/PUMA signaling

TRAIL has been demonstrated to play a critical role in the apoptosis of colorectal cancer (CRC) cells, but drug resistance markedly restricts its therapeutic effects. Objectives: This study aims to investigate whether encorafenib can enhance TRAIL-induced apoptosis of colorectal cancer cells and the underlying mechanism. TRAIL was first used to induce CRC cells. CCK-8 assays were conducted for detecting cell viability of TRAIL-induced CRC cells with encorafenib treatment. Flow cytometry was used to detect the cell apoptosis of CRC cells and western blot was used to measure the expressions of apoptosis-related proteins. The expressions of DR4, DR5, p53, and PUMA were then evaluated by qPCR and western blot. After transfecting the interference plasmid of p53 into CRC cells, the expressions of PUMA and DR5 were further explored. TRAIL reduced the cell viability of CRC cells, and the inhibition was further reinforced under co-treatment of TRAIL and encorafenib. Encorafenib also triggered the promotion of CRC cell apoptosis induced by TRAIL. It was also found that encorafenib exerted its promoting effects on cell apoptosis of CRC cells via the elevation of DR5. Besides, encorafenib administration promoted the expression levels of p53 and PUMA in TRAIL-induced CRC cells. Furthermore, p53 knockdown attenuated the expression of PUMA and DR5 in TRAIL-induced CRC cells treated with encorafenib. This study indicates that encorafenib stimulates TRAIL-induced apoptosis of CRC cells dependent on p53/PUMA signaling, which may provide instructions for the treatment of CRC.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.     

CCN1 induces apoptosis in esophageal adenocarcinoma through p53-dependent downregulation of survivin

Many cancer drugs have been developed to control tumor growth by inducing cancer cell apoptosis. However, several intracellular barriers could fail this attempt. One of these barrier is high expression of survivin. Survivin can interfere caspase activation and thereby abort apoptosis. In this study, we found that CCN1 suppressed the survivin expression in tumor cells of esophageal adenocarcinoma (EAC) and thus allowed apoptosis to finish. Furthermore, we demonstrated that this downregulation was dependent on p53 phosphorylation at Ser20, and CCN1 induced EAC cell apoptosis through the activation of p53.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.