beta-Elemene, a novel plant-derived antineoplastic agent, increases cisplatin chemosensitivity of lung tumor cells by triggering apoptosis.

beta-Elemene, a new plant-derived anticancer agent with low toxicity, has been reported to be effective in the treatment of leukemia and solid tumors. In the current study, we explored the therapeutic application of beta-elemene in sensitizing lung cancer cells to cisplatin. beta-Elemene considerably enhanced the inhibitory effect of cisplatin on cell proliferation in a time- and dose-dependent manner in the human non-small cell lung cancer (NSCLC) cell lines H460 and A549. Furthermore, this effect of beta-elemene on cisplatin activity occurred through the induction of apoptosis in NSCLC cells, as assessed by an ELISA-based assay, TUNEL assay and annexin V binding assay. Consistent with these results, the protein levels of Bax and phospho-Bcl-2 increased and those of Bcl-2 and XIAP decreased in cells treated with beta-elemene in combination with cisplatin, compared with the levels in cells treated with either agent alone. Finally, beta-elemene augmented the cisplatin-induced increases in caspase-3, -7, -9 and -10 activities and cleaved caspase-3, -9 and poly(ADP-ribose) polymerase levels in NSCLC cells. These observations suggest that beta-elemene sensitizes NSCLC cells to cisplatin via a mitochondria-mediated intrinsic apoptosis pathway involving Bcl-2 family proteins and IAPs (inhibitor of apoptosis proteins). Our data provide a rationale for developing a combination of beta-elemene and cisplatin as a regimen for the treatment of lung carcinoma and other cisplatin-resistant tumors.

The human WW45 protein enhances MST1-mediated apoptosis in vivo.

Mammalian sterile 20-like kinase 1 (MST1) is a serine/threonine protein kinase that is activated in response to a variety of apoptotic stimuli and causes apoptosis when over-expressed in mammalian cells. The physiological regulation and cellular targets of MST1 are not well understood. Using a yeast two-hybrid system, we identified human WW45 (hWW45, also called hSav1) as an MST1-binding protein. The association between the two proteins was confirmed by immunofluorescence and co-immunoprecipitation, and hWW45 was present in both the cytoplasm and nucleus. When hWW45 alone was over-expressed, it weakly induced apoptosis. However, hWW45 augmented MST1-induced apoptosis when the two were co-expressed. Conversely, RNA interference-mediated depletion of endogenous hWW45 suppressed MST1-induced apoptosis. These results indicate that hWW45 is required to enhance MST1-mediated apoptosis in vivo and thus is a critical player in an MST1-driven cell death signaling pathway.

Transcription factor E2F-1 is upregulated in human gastric cancer tissues and its overexpression suppresses gastric tumor cell proliferation.

The E2F family members play a critical role in cell cycle regulation and other biological processes in the cell. To better understand the involvement of E2F-1 in the development and progression of gastric tumors, we investigated the mutation and expression of E2F-1 in human gastric cancer tissues and the effect of E2F-1 overexpression on the proliferation of gastric carcinoma cells. In this study, 80 pairs of gastric cancer specimens and paratumor tissues from different patients and 40 stomach mucosa specimens from healthy individuals were examined. PCR-SSCP analysis demonstrated that mutations were not detected in any of the gastric cancer and normal tissue specimens. In addition, the results of an immunohistochemistry assay revealed higher expression rates of E2F-1 (P<0.01) in gastric cancer tissues (72.5%) than in paratumor tissues (30.0%) of the same individuals and stomach mucosa from healthy individuals (22.5%). However, no correlation was observed between the E2F-1 levels and patients' clinical features, such as sex, age, histological types, lymph node metastasis, and clinical stages (P>0.05). Finally, the influence of E2F-1 overexpression on the growth of human gastric carcinoma MKN-45 cells in vitro was assessed by measuring colony formation, cell survival, and cell cycle progression. Our data clearly showed that cell growth and proliferation were significantly inhibited in MKN-45 tumor cells transfected with the expression vector encoding E2F-1 in comparison with nontransfected cells or cells transfected with empty vector. These findings suggest that E2F-1, a stable and conservative gene during the oncogenesis and progression of stomach cancers, may potentially serve as a biomarker for clinical diagnosis of gastric carcinomas and as a target for the development of novel therapeutic interventions to treat this disease.

In vitro combination characterization of the new anticancer plant drug beta-elemene with taxanes against human lung carcinoma.

Beta-elemene has recently raised interest in P.R. China as a novel antitumor plant drug isolated from the Chinese medicinal herb Zedoary. To explore potentially useful combinations of beta-elemene with taxanes in the clinic, we characterized the effects of beta-elemene combined with taxanes in human lung cancer cells using a median effect analysis, micronucleus assay, apoptotic detection, and determination of gene expression in the signaling pathways of apoptosis. The synergistic analysis indicated that the interactions of beta-elemene with paclitaxel or docetaxel ranged from slight synergism to synergism. Combinations of beta-elemene with docetaxel induced much stronger synergistic interactions in p53 mutant H23 cells and p53 null H358 cells than in p53 wild-type H460 and A549 cells. Similar synergistic interactions were observed by micronucleus assay, apoptotic detection, and determination of apoptotic gene expression. Our findings indicate that the synergistic effects achieved with combinations of beta-elemene and taxanes are related to the augmented cytotoxic efficacy of taxanes owing to the action of beta-elemene. In H460 and A549 cells, dose-dependent upregulation of p53 protein expression was observed in cultures treated with docetaxel alone and with docetaxel plus beta-elemene, whereas no significant change in p53 expression was observed in any of the treatment groups in H23 cells. Fas revealed no alteration of expression with any of the treatments in this study. However, the combination treatments induced increased cytochrome c release from mitochondria, significant caspase-8 and -3 cleavage, and downregulation of Bcl-2 and Bcl-XL expression. These results suggest that, although p53 plays an important role in taxane-induced cell death, apoptosis induced by beta-elemene or in combination with docetaxel thereof seems to be initiated through a p53- and Fas-independent pathway via mitochondria in our lung cancer cells. The suppression of specific 'survival' gene expression appears to be the key action leading to the synergistic effect of combination treatments with beta-elemene and taxanes. Finally, the beta-elemene-induced alteration of cell membrane permeability, which has potential to result in enhanced cellular uptake of taxanes, may also contribute to the synergistic interactions of the combination treatments.

Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells.

Apoptosis results in cell death within 10 min after initiation by Bcl-2 family proteins and mitochondria; however, cells enter the apoptotic pathway at different elapsed times after being triggered. Intrinsic factors related to chemical or physical cell damage can initiate apoptosis at a specific cell cycle phase; it is not clear whether cells insulted via an extrinsic pathway also die at a specific cell cycle phase, or how apoptosis is related to cell cycle progression in cells. To illustrate the kinetic changes of apoptosis during cell cycle progression, we examined both intrinsically and extrinsically induced apoptosis in MOLT-4 and Jurkat lymphocytic leukemia cells and in cultured peripheral blood lymphocytes (PBLs) using a recently modified annexin V and propidium iodide method, which detects cell cycle-specific apoptosis. Apoptosis predominantly occurred at a specific cell cycle phase. Leukemia cells were sensitive to induction by both intrinsic (X-rays, UV light, camptothecin, arsenic trioxide, and the traditional Chinese medicine Jinke, which is an extract of Auricularia auricula) and extrinsic factors (via Fas and TNF receptor pathways). The phase at which leukemia cells entered apoptosis depended on the nature of the insult (X-ray or UV, G1-phase; camptothecin, S-phase; arsenic, G1/S phases; Jinke, G1/S phases; and TNF or Fas ligand, G1/S phases), whereas PBLs did not exhibit such insult-dependent differences. PHA-stimulated PBLs entered apoptosis, and additional cells were recruited following additional insults. Unstimulated PBLs remained unresponsive to apoptosis, and proliferating cells became insensitive to the insults after the cell cycle checkpoint was abolished by caffeine. Confluent or starving PBLs were also unresponsive to apoptotic triggers. Thus, apoptotic cell death is a cell cycle event with most, if not all, apoptosis being initiated during a particular cell cycle phase, and changes in the cell cycle result in changes in the apoptotic pattern and schedule. The coordination of apoptosis and proliferation in cells offers a mechanism for the integration of both cell cycle and apoptotic signals.

Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts.

Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.

Dual regulation of soluble tumor necrosis factor-alpha induced activation of human monocytic cells via modulating transmembrane TNF-alpha-mediated 'reverse signaling'.

Transmembrane tumor necrosis factor-alpha (mTNF-alpha) is known to be the precursor of soluble TNF-alpha (sTNF-alpha). mTNF-alpha can act as a ligand on the TNF receptor- (TNFR)- bearing cell through 'forward signaling' or as a receptor on the TNF producing cell through 'reverse signaling'. In the current study, we investigated the role of mTNF-alpha-mediated reverse signaling in regulating sTNF-alpha-induced activation of human monocytic U937 cells. We demonstrated that pretreatment with sTNFRI, for inducing reverse signaling through mTNF-alpha, sensitized U937 cells to sTNF-alpha stimulation, as evidenced by an increase in reactive oxygen production and mRNA levels of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-8) in these cells. Further experiments revealed that IkappaB-alpha degradation was increased in the monocytic cells primed with sTNFRI, implying that reverse signaling of mTNF-alpha sensitizes U937 cells via an NF-kappaB-dependent mechanism. On the other hand, binding of sTNFRI to mTNF-alpha after sTNF-alpha-induced activation of U937 cells reduced mRNA stability (half-life) of IL-1beta and IL-8. The involvement of reverse signaling in the process was verified by using a mutated form of mTNF-alpha lacking the majority of the cytoplasmic domain. Our results clearly showed that enhanced mRNA degradation of the cytokines occurred only in U937 cells transfected with a wild-type mTNF-alpha, but not in those cells transfected with the mutant mTNF-alpha. Taken together, these data suggest that reverse signaling through mTNF-alpha may exert a double role in modulating sTNF-alpha bioactivity. It is positive when reverse signaling occurs prior to sTNF-alpha stimulation, while it is negative when reverse signaling occurs after the sTNF-alpha signal. Thus, our findings strengthen a role of mTNF-alpha-mediated reverse signaling in the regulation of immune-inflammatory response and control of inflammatory reaction.

Antisense telomerase RNA inhibits the growth of human glioma cells in vitro and in vivo.

Telomerase is implicated in the development of cellular immortality and oncogenesis. It has been shown that telomerase activity is considerably higher in the tissue of many different cancers than in normal tissue, and that the inhibition or downregulation of telomerase activity can prevent the malignant proliferation of tumor cells. Antisense oligonucleotides have been widely used in suppressing the expression of genes and, therefore, in the present research, we evaluated the effect of antisense human telomerase RNA (hTR) on glioma cell growth in vitro and in vivo. We showed that antisense hTR cDNA significantly inhibited TJ905 human glioma cell proliferation in vitro and tumor growth in vivo, as determined by MTT assay and by measuring the volume of glioma in nude mice. Consistent with these results, we found that telomerase activity and the mRNA levels of hTR and hTERT (human telomerase reverse transcriptase) expression were markedly decreased in tumor cells treated with antisense hTR cDNA, as assessed by TRAP (telomeric repeat amplification protocol) assay and RT-PCR (reverse transcription-polymerase chain reaction) analysis. Our study conclusively demonstrates that antisense hTR effectively inhibits the growth of human glioma cells in vitro and in vivo and, thus, may be potentially used for gene therapy of malignant gliomas and other cancers.

CD4+ T cells and the Th1/Th2 imbalance are implicated in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is considered to be an organ-specific autoimmune disease. However, the pathogenesis of GO is incompletely understood at the present time. To clarify the immunological differences between newly diagnosed GO and Graves' disease (GD) without ophthalmopathy or healthy controls (HC), we examined T-cell profile and the Th1/Th2 profile cell balance in GO (n=20), GD (n=20) and HC (n=20) using flow cytometry. We also assessed the influence of methimazole on the immunocyte profiles in patients with GO and GD and analyzed the relationship of the immunologic changes with CAS, FT3, FT4, TRAb, TMA and TGA among the three investigated groups. We report in this study that: 1) The percentage of CD4+ T cells and the ratio of CD4+/CD8+ cells were higher, but the population of CD8+ T cells was lower in both GO and GD than those of HC (P<0.05); 2) The percentage of CD8-/IFNgamma+ T cells (Th1) and the ratio of CD8-/IFNgamma+ to CD8-/IL-4+ T cells (Th1/Th2) in GO were considerably higher as compared to those in GD and HC (P<0.05). On the contrary, the population of Th1 cells, as well as the ratio of Th1/Th2 cells, was lower in GD than that of GO and HC (P<0.05); 3) There were no significant differences in T-cell profile and the Th1/Th2 cell balance in either GO or GD patients before and after methimazole treatment; 4) There was a positive correlation of Th1 cell percentage and the Th1/Th2 cell ratio with the clinical activity score (CAS) in GO (P<0.05), whereas CAS in GO had no correlation with the T-cell profile, the percentage of Th2 cells, and TRAb (P>0.05); 5) T-cell subset and the ratio of Th1/Th2 cells did not correlate significantly with FT3, FT4, TRAb, TMA, or TGA in GO and GD (P>0.05). Finally, 6) there were no statistical differences in TRAb, TMA, and TGA between early GO and GD without ophthalmopathy (P>0.05). Collectively, these results indicate that the balance of Th1/Th2 in GO shifts to Th1 dominance and that the cellular immune responses mediated by the Th1-type CD4+ cells might play a dominant role in the pathogenesis of GO, and thus suggest that the Th1 cell percentage and the ratio of Th1/Th2 cell subsets may be potentially utilized as clinical parameters for disease activity, for monitoring the effectiveness of immunosuppressive treatment, or for developing immunospecific forms of therapy for Graves' ophthalmopathy.

Use of bacteriophage in the treatment of experimental animal bacteremia from imipenem-resistant Pseudomonas aeruginosa.

The emergence of antibiotic-resistant bacterial strains still remains a significant problem for antimicrobial chemotherapy in the clinic. Bacterial viruses (bacteriophages or phages) have been suggested to be used as alternative therapeutic agents for bacterial infections. However, the efficacy of phage therapy in treating drug-resistant infections in humans is uncertain. Therefore in the present study, we examined the effectiveness of phages in the treatment of imipenem-resistant Pseudomonas aeruginosa (IMPR-Pa) infection in an experimental mouse model. Twenty-nine strains of phage were isolated from our hospital sewage, and phage ØA392 was representatively used for all testing because it has lytic activity against a wide range of clinical isolates of IMPR-Pa. We found that intraperitoneal (i.p.) injections of one IMPR-Pa strain (3 x 10(7) CFU) caused bacteremia and all mice died within 24 h. A single i.p. inoculation of the phage strain (MOI > or =0.01) at up to 60 min after the bacterial challenge was sufficient to rescue 100% of the animals. This lifesaving effect coincided with the rapid appearance of ØA392 in the circulation (within 2 h after i.p. injection), which remained at substantially higher levels for up to 48 h until the bacteria were eradicated. However, the survival rates of the mice dropped to approximately 50% and 20% when the same dose of this purified phage preparation was administered at 180 min and 360 min, respectively, after IMPR-Pa infections. In addition, we demonstrated that the ability of this phage to rescue bacteremic animals was due to the functional capabilities of the phage and not to a non-specific immune effect. The protection from death occurred only in animals inoculated with bacteria-specific virulent phage strains. When the heat-inactivated phages were used, the survival rate of the infected mice was dramatically reduced to 20% or lower. Moreover, the levels of the antibody against the phage were not significantly changed at the time when the bacteremic animals were protected by the active phages. Finally, our observations revealed that the inoculation of the mice with high-doses of ØA392 alone produced no adverse effects attributable to the phage. These data indicate that phages can save animals from pernicious P. aeruginosa infections and suggest that phage therapy may be potentially used as a stand-alone therapy for patients with IMPR-Pa infections.

Therapeutic effectiveness of bacteriophages in the rescue of mice with extended spectrum beta-lactamase-producing Escherichia coli bacteremia.

The emergence of multidrug-resistant bacteria has become a global crisis. Accumulating evidence shows that bacteriophages (phages) can rescue animals from a variety of lethal infections and be effective in treating drug-resistant infections in humans. Enterobacteriaceae, producing extended spectrum beta-lactamase enzymes (ESBLs), are resistant to a broad range of beta-lactamase antibiotics. One of the most common ESBL-producing gram-negative bacilli in Enterobacteriaceae is Escherichia coli. Since ESBL-producing E. coli poses a formidable challenge in the management of critically ill patients with bacterial infections, we undertook this study to explore the possible therapeutic utility of phages to control ESBL-producing E. coli infections. The phage Ø9882 used in this study was isolated from our hospital sewage and has lytic activity against a broad range of clinical isolates of ESBL-producing E. coli. ESBL-producing E. coli strains (n=30) were isolated in the clinic, and one of them was used to induce bacteremia in a murine model. Bacteremia was established by intraperitoneal (i.p.) injection of 3 x 10(7) CFU/ml, the minimum lethal dose (MLD) of bacterium in this animal model. Mice infected with the MLD of this strain alone died within 14 h, whereas a single i.p. inoculation of Ø9882 (MOI > or =10(-4)) given 40 min after the bacterial challenge led to 100% survival at 24-168 h, compared to 0% survival of saline-treated controls. Protection was obtained even when administration of the phage was delayed up to 60 min after the bacterial infection and the survival rate of infected animals was 60% at 168 h. Furthermore, it was shown that the therapeutic efficacy of Ø9882 in lethal systemic infection in our model is due to the functional capability of the phage and not the nonspecific immune effects. Our data both in vitro and in vivo revealed that: i) the protection of mice from death occurred only in animals infected with selected bacterial strains and the virulent phage specific to them; ii) when the phages were heat-inactivated, survival of the infected mice was strikingly decreased to 0; and iii) the level of antibody against the phage was not substantially elevated when the bacteremic animals were protected by the phage. The present findings indicate that phages can effectively rescue our mouse model from bacteremia and death, and thus provide the rationale and framework to evaluate the therapeutical efficacy of lytic phages against fatal ESBL-producing E. coli infections in humans.

Differential expression of cyclins A, B1, D3 and E in G1 phase of the cell cycle between the synchronized and asynchronously growing MOLT-4 cells.

The use of 'double-thymidine block' was the first widely accepted method for inducing cell synchrony and remains one of the most effective and frequently used techniques for analyzing the cell cycle today. While thymidine is in itself an inhibitor of DNA replication, thymidine blocks are typically used to generate cell synchrony at the G1/S boundary. We have previously presented the first evidence that shows the growth imbalance and altered expression levels of cyclins A, B1, D3 and E in MOLT-4 cells synchronized in the cell cycle by thymidine. The major objective of the present study was to compare the levels of cyclins A, B1, D3 and E in G1 phase of the cell cycle between synchronized and unperturbed asynchronously growing human lymphocyte leukemia MOLT-4 cells. Here, we demonstrate that the sorted, asynchronously growing MOLT-4 cells had considerably lower levels of cyclins A, B1, D3 and E than their counterparts of the cells arrested in G1/S phase, as assessed by flow cytometry. In addition, we confirmed these results by using post-sorting Western blotting, a new method we recently developed for examining protein expression in specific phases of sorted, synchronized or asynchronously growing cells. Our findings revealed that the levels of cyclins D3 and E in the asynchronously growing MOLT-4 cells were significantly lower than those in synchronized cultures. Interestingly, protein expression levels of cyclins A and B1 in the asynchronously growing MOLT-4 cells were barely measurable, suggesting that these proteins were either not expressed or under detectable levels. These studies indicate that our synchronization protocol may have disturbed cell proliferation and metabolism as evidenced by significant differences in the expression of cyclins between asynchronously growing and synchronized cells, and further suggest that the levels of cyclins A, B1, D3 and E in synchronized cultures cannot represent those in unperturbed, asynchronously growing cells. Thus, it appears that thymidine-treated, synchronized cells may not be suitable experimental models for the study of normal cell cycle.

Subcellular localization of caspase-3 activation correlates with changes in apoptotic morphology in MOLT-4 leukemia cells exposed to X-ray irradiation.

Caspase-3 is a critical effector caspase for apoptosis, which cleaves proteins, including cytoskeletal and associated proteins, kinases, and members of the Bcl-2 family of apoptosis-related proteins. This leads to changes in apoptotic morphology, such as membrane externalization and cytoplasm and nuclear condensation. It has been reported that pro-caspase-3 is activated in the cytosol. However, it remains obscure how caspase-3 activation correlates to serial changes in cell morphology during apoptosis. The current study was therefore undertaken to assess the relationship between caspase-3 activation and its subcellular localization and alterations in apoptotic morphology in MOLT-4 human leukemia cells exposed to X-ray irradiation. Fluorescence labeled inhibitor of caspases (FLICA) was used to detect caspase-3 activity in apoptotic cells in this project; cell morphology and caspase-3 sub-localization were determined by confocal microscopy. Our data showed that MOLT-4 cells presented typical morphological changes in apoptosis, such as membrane reversion, DNA fragmentation, and formation of apoptotic cell bodies following 10 Gray (Gy) of X-ray irradiation. Caspase-3 was activated 2 h after X-ray irradiation, and its activity increased markedly after 4-6-h exposure. Membrane reversion in MOLT-4 leukemia cells was detected by Annexin V assay at 4 h following X-ray irradiation, 2 h after the elevated caspase-3 activity was measured. Cytologically, activation of caspase-3 was first observed close to the inside surface of the cellular membrane, then transferred to the cytoplasm, and finally translocated to the nuclear region. We conclude that caspase-3 is activated in MOLT-4 cells following exposure to X-rays, and that the enhanced caspase-3 activity and its sub-localization shifting is correlated to changes in apoptotic morphology. The spatial shift of activated caspase-3 in X-ray-induced apoptotic MOLT-4 leukemia cells is a process of crucial importance for apoptosis.

Molecular mechanism of antitumor activity of taxanes in lung cancer (Review).

Lung cancer is the most common cause of cancer mortality in both male and female patients in the United States of America, as well as in the rest of the world. Over one million people are diagnosed with lung cancer every year worldwide. The taxane is one of the most powerful classes of novel antitumor agents and has become an integral part of several commonly used chemotherapy regimens in lung cancer management over the past few years. Although the ability of taxanes to disrupt microtubule dynamics is well documented, the molecular basis by which taxanes suppress cancer cell growth and induce apoptotic cell death is not clearly defined. In this review, we focus on the molecular mechanisms of the antitumor activity of taxanes (paclitaxel and docetaxel) in lung cancer, and discuss the interactions of taxanes with microtubules, the roles of cell cycle control and cell death induction in the anticancer action of taxanes, as well as the signal transduction pathways involved in the processes. In addition, we discuss the possible mechanisms of taxane resistance, because drug resistance to these anti-neoplastic agents affects therapy efficacy and is also a major obstacle in the clinic for the successful treatment of lung cancer. Understanding the molecular mechanisms underlying the antitumor effect of taxanes and the drug resistance to taxanes may lead to the design of biologically and pharmacologically targeted therapeutic strategies for taxane resistant tumors, and to the improvement of chemotherapy effect and cancer patient survival.

Overexpression of the N-terminal end of the p55gamma regulatory subunit of phosphatidylinositol 3-kinase blocks cell cycle progression in gastric carcinoma cells.

pRb and its family members p130 and p107 regulate cell cycle progression and direct G1/S transition in mammalian cells through interaction with the transcription factor E2F. Phosphatidylinositol 3-kinase (PI3K) is an essential component of growth factor-regulated pathways and plays a crucial role in the regulation of cellular proliferation and differentiation. It has been demonstrated that PI3K can regulate cell cycle progression via Akt-mediated pathway. However, the possible interactions between PI3K and Rb pathways remain to be defined. It was reported that the unique 24-amino-acid N-terminal end of the p55 regulatory subunits of PI3K is an Rb-binding domain and affects Rb action or Rb-E2F interaction. The 24 N-terminal amino acids of p55gamma encoded by a cDNA construct could compete with the endogenous p55gamma for binding to Rb, which influences Rb-mediated signaling and blocks cell cycle progression. In the current study, we investigated the effects of this 24-peptide on cell proliferation in human gastric carcinoma MKN-28 cells by means of cell cycle analysis, BrdU incorporation, and determining the levels of cell cycle regulatory molecule expression. Our results showed that p55gammaPI3K and the Rb family members p130 and p107 exist in MKN-28 cells, while the p110PI3K was not detected. Moreover, p55gammaPI3K was found binding to p130/p107 in these cells. We demonstrated that the introduction of the plasmid N24p55-GFP (harboring the cDNA for the 24 N-terminal amino acids of p55gamma) into MKN-28 cells caused cell cycle arrest at G1. Furthermore, we showed that the over-expression of the 24-peptide in MKN-28 cells decreased the population of cells incorporating BrdU and reduced the levels of cyclin D1 and cyclin A. These observations suggest that PI3K can regulate cell cycle progression and cell proliferation in human gastric tumor cells via Rb-mediated pathway, and that this effect of PI3K is mediated through a direct association with Rb via the N-terminal end of its p55 kDa regulatory subunits and modulating Rb-E2F interactions. Taken together with previous studies, our data provide a new therapeutic target in human stomach cancer. Strategies targeting PI3K signal transduction or the association of PI3K with Rb, or regulating PI3K-Rb interactions could be employed for gene therapy or chemotherapy of gastric cancer and other tumors.

Demonstration of differential gene expression between sensitive and resistant ovarian tumor cells by fluorescence differential display-PCR analysis.

Chemotherapy plays a major role in cancer management; however, acquired drug resistance remains a significant problem for ovarian cancer treatment. Chemoresistance is regulated by the coordinated expression of a set of genes. Thus, the identification of genes specifically modulated in the process provides an important step toward the discovery of underlying molecular mechanisms in drug resistance events. We recently developed five drug-resistant human ovarian carcinoma cell lines, including two cisplatin (cis) resistant cell lines, two carboplatin (car) resistant cell lines and one taxol (tax) resistant cell line. In this study, we investigated differential gene expression between these resistant cell lines and their parental cell lines by the fluorescence differential display-polymerase chain reaction (FDD-PCR) technique. We first screened and identified differentially expressed genes in the resistant ovarian cancer cell lines, and we then sequenced and analyzed these genes by bioinformatics software. A total of 33 fragments were displayed in the two resistant cell lines (S-cis and S-car) derived from the sensitive SKOV-3 cell line, and 36 fragments were displayed in the three resistant cell lines (A-cis, A-car and A-tax) derived from the sensitive A2780 cell line. After purification, cloning, sequencing, and homology analysis on the NCBI BLAST GenBank, 12 gene fragments were identified from the resistant S-cis and S-car cells, and 23 gene fragments were identified from the resistant A-cis, A-car and A-tax cells. Although a homolog search of the NIH GenBank revealed that most of the gene fragments were not significantly associated with the known drug resistance-related genes, our study conclusively demonstrates that FDD-PCR is a useful tool for analyzing the differential gene expression between resistant and sensitive tumor cells and for identifying novel chemoresistance-associated genes and potential biological markers or genetic markers of drug resistance.

Characterization of the mechanisms of electrochemotherapy in an in vitro model for human cervical cancer.

Electrochemical treatment is among the most effective therapies in the management of cervical malignancy. However, the mechanism of action of this treatment remains largely unknown. Therefore, the purpose of the current project was to establish a suitable eletrochemotherapy regimen for cervical cancer and to investigate the mechanism of the therapy in an in vitro model for human cervical carcinoma. HeLa cells were used as a model for cervical cancer in this study, and the effect of electrochemical treatment on these cells was examined in four different dosage groups (5 V + 5 C, 10 V + 5 C, 5 V + 10 C and 10 V + 10 C). Our results showed that the combinations of lower voltage and higher current (5 V/10 V + 10 C) had a greater anticancer effect in this model as compared to other groups. In addition, we compared the cytotoxic effect between electrochemical treatment and different pH condition treatments in this system, and found that the efficacy of electrochemical treatment in cell killing was better than that of acidic or basic medium treatment. Moreover, we demonstrated that the efficacy of electrochemical treatment was correlated with the degree of ionization and alteration in pH scale. The electrodes were basic on the cathode side which elevated the cations K+, Ca2+ and Mg2+, while the electrodes were acidic on the anode side which reduced the anion Cl-. We also assessed the effect of electrochemical treatment on cell cycle distribution in HeLa cells and showed that the percentage of cells in the G1 phase of the cell cycle was increased (G1 arrest), while the cell population in the S phase was decreased. Furthermore, we demonstrated that the levels of the cell cycle regulator cyclin D1 expression were dramatically reduced when 5 V/10 V + 10 C treatments were applied to these cells, as determined by RT-PCR analysis. By contrast, no significant changes in the levels of cyclin B1, CDK1 or CDK4 were detected. Based on these observations, we conclude that the combination of lower voltage and higher current may be a potentially effective eletrochemotherapy regimen for cervical cancer in the clinic, and that the antitumor effect of electrochemical treatment on cervical carcinoma cells is mediated partly via regulating ionization degree, pH state and cell cycle control.

Down-regulation of cyclin E expression by caffeine promotes cancer cell entry into the S-phase of the cell cycle.

Expression of cyclin E is believed to be a critical factor promoting cell entry into the S-phase and cell proliferation. Indeed, normal proliferating cells and most tumor cell lines are characterized by the existence of a minimal cyclin E threshold level in the G1-phase, and only those cells expressing cyclin E over this threshold enter into the S-phase of the cell cycle. However, through studying clinical tumor tissue specimens, we recently observed that some cancer cells can enter into the S-phase with minimal levels of cyclin E expression. In an effort to establish an in vitro cell model system for studying the mechanisms underlying this phenomenon, we treated MOLT-4 lymphocyte leukemia cells with 50 mM caffeine and found that the levels of cyclin E expression were decreased markedly in these cells following 2 to 4-h exposure to caffeine. Quite unexpectedly, we observed that the percentage of the cells progressing through the S-phase increased despite the reduced levels of cyclin E, as analyzed for the cellular DNA contents, expression of nuclear-bound PCNA, immunolabelling with Ki-67 antibody and incorporation of BrdU. In fact, these cells entered into the S-phase with a level of cyclin E well below the threshold level for untreated cells, thus suggesting that lower levels of cyclin E expression are associated with cell proliferation under certain circumstances. We speculate that caffeine may enhance MOLT-4 cell entrance into the S-phase through activation of Cdc25, which in turn activates cyclin-dependent protein kinases (CDKs) including CDK2 and drives the cell cycle progression; while degradation of cyclin E by the ubiquitin/proteasome pathway may account for the decreased levels of cyclin E in these cells. Our findings from both the MOLT-4 cell line and patients' cancer tissues may help decipher the mystery of the deregulation of cell cycle progression and carcinogenesis in some malignant tumors.

Molecular basis of cellular response to cisplatin chemotherapy in non-small cell lung cancer (Review).

Cisplatin is one of the most potent anticancer agents, displaying significant clinical activity against a variety of solid tumors. For more than two decades, the most effective systemic chemotherapy for non-small cell lung cancer (NSCLC), the leading cause of cancer morbidity and mortality among men and women in the western world, was cisplatin-based combination treatment. Unfortunately, the outcome of cisplatin therapy on NSCLC seems to have reached a plateau. Therefore, the biological mechanisms of cisplatin action need to be understood in order to overcome the treatment plateau on NSCLC. Moreover, the development of resistance is a hurdle in the use of this drug. The molecular mechanisms that underlie this chemoresistance are largely unknown. Possible mechanisms of acquired resistance to cisplatin include reduced intracellular accumulation of cisplatin, enhanced drug inactivation by metallothionine and glutathione, increased repair activity of DNA damage, and altered expression of oncogenes and regulatory proteins. In addition, it is generally accepted that cytotoxicity of cisplatin is mediated through induction of apoptosis and arrest of cell cycle resulting from its interaction with DNA, such as the formation of cisplatin-DNA adducts, which activates multiple signaling pathways, including those involving p53, Bcl-2 family, caspases, cyclins, CDKs, pRb, PKC, MAPK and PI3K/Akt. Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may contribute to the inability of cells to detect DNA damage or to induce apoptosis. Towards an understanding of the molecular basis of the cellular response to cisplatin-based chemotherapy in NSCLC, in this review we provide some insights into the pathways involved in cisplatin damage from entering the cells to execution of apoptosis or survival of NSCLC cells. We believe that as more and more molecular mechanisms of response to cisplatin-based therapy are unraveled, this knowledge should provide a basis for further studies to improve our understanding of molecular events associated with lung NSCLC as well as to devise novel and effective therapeutic approaches to overcome the treatment plateau or reverse drug resistance in this disease.

Correlation of serum VEGF levels with clinical stage, therapy efficacy, tumor metastasis and patient survival in ovarian cancer.

Vascular endothelial growth factor (VEGF) has been shown to play an important role in tumor growth and progression. However, the clinical implications of VEGF expression in ovarian tumors are not fully understood. We therefore investigated the serum level of VEGF in patients with ovarian tumors and explored the potential use of VEGF as a tumor marker for diagnosis, treatment and prognosis of human ovarian cancer. The serum VEGF (sVEGF) levels in 120 patients with ovarian carcinoma, 25 patients with benign ovarian tumor and 90 healthy female blood donors were measured by an enzyme-linked immunosorbent assay in this study. We also determined the levels of sVEGF in patients with epithelial ovarian cancer before and after surgery. Our results showed that: (i) ovarian cancer patients had significantly higher levels of sVEGF compared to those of patients with benign ovarian tumor or those of healthy individuals. As a cut-off at 100 pg/ml, the sensitivity and specificity of sVEGF levels for diagnosing ovarian carcinoma were 77.1% and 87%, respectively. (ii) sVEGF levels were markedly elevated in patients with advanced stage or poorly-differentiated ovarian cancer, as well as in those with more ascites (>500 ml), as compared to patients with early stage and well-differentiated ovarian cancer, or those with less ascites (<500 ml). However, there was no significant difference in sVEGF levels among different pathological subtypes of ovarian carcinoma. (iii) The post-operative sVEGF levels were significantly lower than the pre-operative sVEGF levels. (iv) We measured significantly higher levels of sVEGF in patients with metastasis as compared to patients lacking metastasis. Lastly (v) the average survival-time in patients with higher levels of sVEGF (>100 pg/ml) was 28 months, while the average survival-time in patients with lower levels of sVEGF (<100 pg/ml) was 35 months, indicating that the elevations in sVEGF level are correlated with patient survival and tumor metastasis in ovarian carcinoma. These data suggest that VEGF may be a useful serological biomarker for clinical diagnosis and prognosis of ovarian cancer, for follow-up of ovarian tumor metastasis and for monitoring the efficacy of therapy in patients with ovarian carcinomas.