Enhanced G1 arrest and apoptosis via MDM4/MDM2 double knockdown and MEK inhibition in wild-type TP53 colon and gastric cancer cells with aberrant KRAS signaling.

Murine double minute homolog 2 (MDM2) is an oncoprotein that induces p53 degradation via ubiquitin-ligase activity. MDM4 cooperates with MDM2-mediated p53 degradation, directly inhibiting p53 transcription by binding to its transactivation domain. Our previous study reported that the simultaneous inhibition of MDM2 and MDM4 using nutlin-3 (an inhibitor of the MDM2-p53 interaction) and chimeric small interfering RNA with DNA-substituted seed arms (named chiMDM2 and chiMDM4) more potently activated p53 than the MDM2 or MDM4 inhibitor alone and synergistically augmented antitumor effects in various types of cancer cells with the wild-type (wt) TP53. Recently, the synergism of MDM2 and mitogen-activated protein kinase kinase (MEK) inhibitors has been demonstrated in wt TP53 colorectal and non-small cell lung cancer cells harboring mutant-type (mt) KRAS. The current study examined whether chiMDM4 augmented the synergistic antitumor effects of MDM2 and MEK inhibition using chiMDM2 or nutlin-3 and trametinib, respectively. ChiMDM2 and trametinib used in combination demonstrated a synergistic antitumor activity in HCT116 and LoVo colon cancer cells, and SNU-1 gastric cancer cells harboring wt TP53 and mt KRAS. Furthermore, chiMDM4 synergistically enhanced this combinational effect. Similar results were observed when nutlin-3 was used instead of chiMDM2. MDM4/MDM2 double knockdown combined with trametinib treatment enhanced G1 arrest and apoptosis induction. This was associated with the accumulation of p53, suppression of phosphorylated-extracellular signal-regulated kinase 2, inhibition of retinoblastoma phosphorylation, suppression of E2F1-activated proteins, and potent activation of pro-apoptotic proteins, such as Fas and p53 upregulated modulator of apoptosis. The results inidcated that the triple inhibition of MDM4, MDM2 and MEK exerted a potent antitumor effect in wt TP53 colon and gastric cancer cells with mt KRAS. Simultaneous activation of p53 and inhibition of aberrant KRAS signaling may be a rational treatment strategy for gastrointestinal tumors.

MDM4 as a Prognostic Factor for Patients With Gastric Cancer With Low Expression of p53.

BACKGROUND/AIM: The oncoproteins murine double minute (MDM) 2 and MDM4 inactivate tumor-suppressor protein p53. Their mutual relationship with the prognosis of gastric cancer (GC) remains unknown. PATIENTS AND METHODS: Expression of MDM2, MDM4, and p53 in tumors of 241 patients with GC were evaluated immunohistochemically. Effects of overexpression of MDM4 on tumor-growth properties and sensitivity to cytotoxic drugs were investigated using NUGC4 human GC cell line. RESULTS: High expression of p53 was associated with poor overall survival in the whole population. Among 173 patients with low expression of p53 (implying nonmutation), high expression of MDM4 was an independent factor of poor prognosis in both stage I-III and IV, but of MDM2 was not. MDM4-transduced NUGC4 cells formed twice as many colonies and had a higher 50% inhibitory concentration for 5-fluorouracil and oxaliplatin than did the control cells. CONCLUSION: MDM4 expression is a factor conferring poor prognosis in patients with GC with low expression of p53 and may confer drug resistance.

Augmented antitumor activity of 5-fluorouracil by double knockdown of MDM4 and MDM2 in colon and gastric cancer cells.

Inactivation of the TP53 tumor suppressor gene is essential during cancer development and progression. Mutations of TP53 are often missense and occur in various human cancers. In some fraction of wild-type (wt) TP53 tumors, p53 is inactivated by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA-modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wt TP53 and high MDM4 expression (wtTP53/highMDM4). In the present study, MDM4/MDM2 double knockdown with the siRNAs enhanced 5-fluorouracil (5-FU)-induced p53 activation, arrested the cell cycle at G1 phase, and potentiated the antitumor effect of 5-FU in wtTP53/highMDM4 human colon (HCT116 and LoVo) and gastric (SNU-1 and NUGC-4) cancer cells. Exposure to 5-FU alone induced MDM2 as well as p21 and PUMA by p53 activation. As p53-MDM2 forms a negative feedback loop, enhancement of the antitumor effect of 5-FU by MDM4/MDM2 double knockdown could be attributed to blocking of the feedback mechanism in addition to direct suppression of these p53 antagonists. Intratumor injection of the MDM4/MDM2 siRNAs suppressed in vivo tumor growth and boosted the antitumor effect of 5-FU in an athymic mouse xenograft model using HCT116 cells. These results suggest that a combination of MDM4/MDM2 knockdown and conventional cytotoxic drugs could be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers.

Antitumor effects of a sirtuin inhibitor, tenovin-6, against gastric cancer cells via death receptor 5 up-regulation.

Up-regulated sirtuin 1 (SIRT1), an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53). Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5). In the KatoIII cell line (TP53-null), DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

MDM4 expression as an indicator of TP53 reactivation by combined targeting of MDM2 and MDM4 in cancer cells without TP53 mutation.

MDM2 and MDM4, a structurally related MDM2 homolog, negatively regulates expression and functions of TP53 tumor suppressor gene. To explore the precise expression patterns and function of MDM2 and MDM4 in wild-type (wt) TP53 cancer cells, we analyzed 11 various cancer cell lines with wt TP53. All cell lines exhibited deregulated expression of MDM2 and MDM4, and were divided into two distinct types; the one expressing high levels of MDM4 and another expressing low levels of MDM4. The low MDM4 type expressed higher MDM2 levels than the high MDM4 type. In cells with high MDM4 expression, knockdown of MDM4 or MDM2 reactivated TP53, and simultaneous knockdown of MDM2 and MDM4 synergistically reactivated TP53. In contrast, in cells with low MDM4 expression, knockdown of only MDM2 reactivated TP53. These results suggest that both MDM2 and MDM4 are closely involved in TP53 inactivation in cancer cells with high MDM4 expression, whereas only MDM2, and not MDM4, is a regulator of TP53 in cells with low MDM4 expression. MDM4 expression in wt TP53-tumors is a potential indicator for TP53 reactivation cancer therapy by simultaneous targeting of MDM4 and MDM2. Specific knockdown of MDM2 and MDM4 might be applicable for TP53 restoration therapy.

Predicting skin toxicity according to EGFR polymorphisms in patients with colorectal cancer receiving antibody against EGFR.

BACKGROUND/AIM: Monoclonal antibodies against epidermal growth factor receptor (EGFR) can extend progression-free survival (PFS) and overall survival (OS) in patients with unresectable colorectal cancer; however, skin toxicity often interferes with therapy continuation. PATIENTS AND METHODS: We analyzed the polymorphisms in EGFR and IgG fragment C receptor (FCGR) genes and determined their associations with clinical outcomes including PFS, OS, and skin toxicity. Five polymorphisms in EGFR and FCGR genes in 32 patients with unresectable colorectal cancer who were treated with antibodies against EGFR were examined. RESULTS: Patients carrying the C/C genotype of the EGFR D994D polymorphism displayed significantly less skin toxicity than those with other genotypes, although no significant differences in PFS and OS were noted and no significant interactions were detected for other gene polymorphisms. CONCLUSION: These results suggest that the EGFR D994D polymorphism is a useful biomarker for predicting the severity of skin toxicity in patients receiving antibody against EGFR.

The sirtuin inhibitor tenovin-6 upregulates death receptor 5 and enhances cytotoxic effects of 5-fluorouracil and oxaliplatin in colon cancer cells.

It has been reported that upregulated SIRT1 (NAD(+)-dependent class III histone deacetylase) deacetylates the p53 protein, represses its function, and allows for tumor cell growth in various cancers. Here we investigated antitumor effects of tenovin-6, a small-molecule inhibitor of SIRT1 and SIRT2, in various colon cancer cell lines. Tenovin-6 induced apoptosis in all five colon cancer cell lines investigated (two cell lines with wild-type p53 and three with mutant p53) regardless of the p53 mutation status. This effect was accompanied by accumulation of death receptor 5 (DR5) in most cell lines. DR5 silencing in HCT116 cells strongly attenuated tenovin-6-induced apoptosis. We investigated the effect of combining tenovin-6 with conventional anticancer agents 5-fluorouracil (5-FU), SN-38 (an active metabolite of irinotecan), and oxaliplatin. Synergistic antitumor effects of tenovin-6 were observed in combination with either 5-FU or oxaliplatin in vitro. The combination of tenovin-6 and oxaliplatin exhibited potent growth inhibition of HCT116 xenograft tumors in vivo. In conclusion, tenovin-6 induced apoptosis in human colon cancer cells through the activation of the DR5 signaling pathway and enhanced the antitumor properties of 5-FU and oxaliplatin. These results may help develop a novel treatment option for colorectal cancer using a SIRT inhibitor.

The E1 protein of human papillomavirus type 16 is dispensable for maintenance replication of the viral genome.

Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16.

Potent in vitro and in vivo antitumor effects of MDM2 inhibitor nutlin-3 in gastric cancer cells.

The tumor suppressor gene p53 is the most frequently mutated gene in human cancers. However, its mutation rate is relatively low in gastric cancer compared with other cancers. In this study, we investigated the mechanisms underlying the antitumor effects of nutlin-3, an inhibitor of human homolog of murine double minute 2 (MDM2). MDM2 is a negative regulator of p53. Four gastric cancer cell lines with wild-type p53 (wt p53) and three with mutant-type p53 (mt p53) were analyzed for MDM2 and MDM4 expression by immunoblotting, and for their gene amplification by quantitative real-time PCR. Moreover, the viability of cells exposed to nutlin-3 was examined by WST-8 assay, and the expression of p53 and its downstream genes was analyzed by immunoblotting. Nutlin-3 stabilized p53 and increased the expression of p21(WAF1) and Noxa, and cleaved poly (ADP)-ribose polymerase regardless of the pre-expression levels of MDM2 and MDM4 in gastric cancer cells with wt p53. Flow cytometry revealed that nutlin-3 arrested the cell cycle in G(1) phase and induced apoptosis in the cell lines. These nutlin-3 effects were not observed in the cell lines with mt p53. Nutlin-3 exerted additive or synergistic cytotoxicity in combination with 5-fluorouracil or cisplatin in most cell lines with wt p53. An in vivo antitumor effect of nutlin-3 alone and its additive augmentation by 5-fluorouracil were confirmed in an MDM2 overexpressed xenograft tumor model. Nutlin-3 showed potent antitumor activity against human gastric cancer cells with wt p53 and shows promise as a single agent and in combination with conventional anticancer drugs.

[Q fever in hospital-acquired pneumonia].

We studied the effects of Q fever in hospital-acquired pneumonia. The subjects consisted of 121 cases with hospital-acquired pneumonia treated during the period from December 2004 till June 2007. Q fever was diagnosed using a PanBio Coxiella burnetii ELISA test kit. There were no patients with acute infection by Coxiella burnetii. It is concluded that C. burnetii cannot induce onset of hospital-acquired pneumonia.

[Clinical effects of intravenous ciprofloxacin on community-acquired pneumonia with positive Immunocard Mycoplasma test results].

We studied the clinical effects of intravenous ciprofloxacin (CPFX) on community-acquired pneumonia in patients with positive Immunocard Mycoplasma test results. The subjects were 35 patients (59.4 +/- 24.8 years old) with community-acquired pneumonia with positive Immunocard Mycoplasma test results. We infused CPFX 300mg copy intravenously twice daily for 3-14 days. It was effective in 33 of 35 patients, with an efficacy rate of 94.3%. Adverse reactions consisted of itching in 2 patients, malaise in 2 patients, drug eruption in 1 patient, elevation of GPT in 1 patient and elevation of BUN in 1 patient, but all were mild. We conclude that intravenous CPFX is useful for community-acquired pneumonia in case with positive Immunocard Mycoplasma test results.

Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect.

Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2-8 from the 5'end of the guide strand; its complementary sequence; the 5'end of the guide strand and the 3'overhang of the passenger strand. However, most part of the 3' two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3'end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA-RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA-RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.

[Study of the cases with multidrug-resistant Pseudomonas aeruginosa by sputum culture].

We evaluated the clinical features of multidrug-resistant Pseudomonas aeruginosa cases determined by sputum culture between April, 2005 and December, 2006. The clinical features of most cases were: (1) pneumonia in the elderly with cerebrovascular diseases, (2) previous administration of carbapenems and antipseudomonal cephems, (3) PIPC, CAZ and ISP sensitve MDRP, (4) MRSA was isolated concurrently, (5) not necessary of therapy against MDRP, (6) good outcome.

[Case of multiple intrapulmonary lymph nodes].

A 45-year-old man who had hypertension, hyperthyroidism, and bronchial asthma was admitted to our hospital because of a low-grade fever and chest pain. The physical findings and laboratory data were almost all within normal limits except for evidence of mild inflammation and liver dysfunction. The chest X-ray findings seemed normal, but a computed tomography (CT) scan showed multiple nodules in both lower lung fields. We suspected the cryptococcosis or lung cancer. Biopsy by video-assisted thoracoscopic surgery (VATS) yielded a diagnosis of multiple intrapulmonary lymph nodes. In cases with the above radiologic findings, careful attention should be paid to making the differential diagnosis between intrapulmonary lymph nodes and primary lung cancer. The promotion of diagnostic imaging and advances in techniques have made it easier to identify small peripheral nodules in the lungs, and we now know of their existence. Solitary intrapulmonary lymph nodes are encountered frequently, but multiple or increasing numbers of nodes, as in our case, are very rare. Moreover, because cases with elevated CEA levels have been reported, differentiation from lung cancer appears to be important.

Myeloperoxidase is a key regulator of oxidative stress mediated apoptosis in myeloid leukemic cells.

PURPOSE: We reported previously that reactive oxygen species (ROS) are key mediators of apoptosis induced by a polyphenol, (-)-epigallocatechin-3-gallate (EGCG), in myeloid leukemic cells. This study aimed to further examine the mechanism of ROS-mediated apoptosis induced by EGCG and its relationship to the heme enzyme myeloperoxidase (MPO). EXPERIMENTAL DESIGN: We established stably transfected K562 cells expressing wild-type and mutant MPO. Then, sensitivity against EGCG and other ROS-inducing agent was examined and further investigated the detailed molecular mechanism of ROS-inducing apoptosis in MPO-positive leukemic cells. RESULTS: EGCG rapidly induced apoptosis in MPO-positive leukemia cells. Preincubation of myeloid leukemic cells with the MPO-specific inhibitor, 4-aminobenzoic acid hydrazide, and the heme biosynthesis inhibitor, succinylacetone, resulted in inhibition of the intracellular MPO activity, ROS production, and induction of apoptosis following addition of EGCG. Overexpression of MPO sensitized EGCG-resistant K562 cells to apoptosis induced by EGCG. In contrast, an enzymatically inactive MPO mutant-expressing K562 cell could not respond to EGCG, suggesting that MPO is important for determining the sensitivity to EGCG-induced oxidative stress. Hypochlorous acid scavengers and the hydroxyl radical (.OH) scavenger inhibited EGCG-induced apoptosis in myeloid leukemic cells. The fluorescence intensity of both aminophenyl fluorescein- and hydroxyphenyl fluorescein-loaded myeloid leukemic cells significantly increased on stimulation with EGCG, indicating that EGCG generated highly toxic ROS in myeloid leukemic cells. CONCLUSIONS: These results indicated that highly toxic ROS such as .OH generated via the hydrogen peroxide/MPO/halide system induce apoptosis and that ROS may be the direct mediators of EGCG-induced apoptosis in MPO-positive leukemic cells.

[Positive phase periods of ImmunoCard Mycoplasma tests].

We evaluated the positive phase period of ImmunoCard Mycoplasma tests. The subjects were 74 penumonia patients (male : 38, female : 36, 17-94 years old) with positive ImmunoCard Mycoplasma tests. ImmunoCard Mycoplasma tests were performed every week for 8 weeks later, then every 4 weeks until negative conversion. The positive phase period was within a week in 30 of 74 patients (40.5%) and within 4 weeks in 52 patients (70.3%). In each generation the positive phase period of the most patients was within a week. The positive phase period of the elderly had no tendency to be longer than that of the young patients. These results indicated that about half of the patients with positive ImmunoCard Mycoplasma tests showed Mycoplasma infection which occurred within the past 1 week.

Coxiella burnetii and acute exacerbations/infections in patients with chronic lung disease.

The aim of the present study was to determine the incidence of Q fever in patients with an acute exacerbation of a chronic lower respiratory tract infection. Eighty patients treated for acute exacerbation of chronic lower respiratory tract infections during a 30-month period were studied. Q fever was diagnosed by ELISA. Two elderly woman with pre-existing bronchiectasis (2.5%) were diagnosed as having an acute infection by Coxiella burnetii. The acute illness was considered to be a result of mixed infection with Pseudomonas aeruginosa and Haemophilus influenzae with C. burnetii. Co-infection with C. burnetii can occur during a bacterial exacerbation of a chronic lower respiratory tract infection.

[Testing for Mycoplasma pneumonia using the ImmunoCard Mycoplasma rapid test].

We evaluated the effectiveness of ImmunoCard Mycoplasma rapid tests in all patients admitted with community-acquired pneumonia (CAP) between January, 2004 and December, 2005. ImmunoCard Mycoplasma rapid tests were performed on the 1st day of admission and we analyzed the frequency of positive cases among CAP cases according to month and age. A total of 82 of 270 (33.7%) and 41 of 257 (16.0%) were positive among CAP cases in 2004 and 2005, respectively. More positive cases were seen between spring and early summer and in cases aged 70 years or more, especially those over 80 years old. These results indicated that further evaluation is required among positive cases in elder group.

Zerumbone, a bioactive sesquiterpene, induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- and mitochondria-mediated pathway.

We demonstrated here for the first time that zerumbone (ZER), a natural cyclic sesquiterpene, significantly suppressed the proliferation of promyelocytic leukemia NB4 cells among several leukemia cell lines, but not human umbilical vein endothelial cells (HUVECs), by inducing G2/M cell cycle arrest followed by apoptosis with 10 microM of IC50. Treatment of NB4 cells with growth-suppressive concentrations of ZER resulted in G2/M cell cycle arrest that was associated with a decline of Cyclin B1 protein, but with the phosphorylation of ATM/ Chk1/Chk2. In addition, ZER induced the phosphorylation of Cdc25C at the Thr48 residue and Cdc2 at the Thr14/Tyr15 residues. Furthermore, ZER-induced apoptosis in NB4 cells was initiated by the expression of Fas (CD95)/Fas Ligand (CD95L), concomitant with the activation of caspase-8. ZER was also found to induce the cleavage of Bid, a mediator that is known to connect the Fas/CD95 cell death receptor to the mitochondrial apoptosis pathway. ZER also induced the cleavage of Bax and Mcl-1 proteins, but not Bcl-2 or Bcl-XL. ZER-induced apoptosis took place in association with a loss of the mitochondrial transmembrane potential as well as the activation of caspase-3 and -9, resulting in the degradation of the proteolytic poly (ADP-ribose) polymerase (PARP). ZER also triggered a release of cytochrome c into the cytoplasm. Both antagonistic anti-Fas antibody ZB4 and pan-caspase inhibitor Z-VAD inhibited ZER-induced apoptosis in NB4 cells. Taken together, ZER is an inducer of apoptosis in leukemic cells that specifically triggers the Fas/CD95- and mitochondria-mediated apoptotic signaling pathway.

p27KIP1 and GATA-1 are potential downstream molecules in activin A-induced differentiation and apoptosis pathways in CML cells.

p27KIP1 is known as a regulator of cellular differentiation and apoptosis in human cancer cells. We have previously reported that human chronic myeloid leukemia (CML) KU812 and K562 cells show inhibited cellular proliferation in response to treatment with activin A, a member of TGF-beta superfamily. Apoptosis and erythroid differentiation can be induced in KU812 and K562 cells, respectively. We report herein that activin A induced the expression of p27KIP1 in CML cells along with the induction of cellular differentiation and apoptosis. There are putative binding sequences of erythroid-related transcription factor GATA-1 in the promoter region of the human p27KIP1 gene. Expression of GATA-1 protein in activin A-treated KU812 and K562 cells showed dissimilar regulation in these two cell lines. Induction of p27KIP1 was commonly observed, but it did not correspond to the expression levels of GATA-1 in either line of activin A-treated CML cells. In addition, ERK protein was rapidly and transiently activated with activin A in both types of CML cells, suggesting that phosphorylation of ERK is required for activin A signaling in CML cells. These results indicate that both p27KIP1 induction and regulation of GATA-1 play essential roles in activin A-induced erythroid differentiation and apoptosis.