Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma.

The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra-terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti-tumour activity of the novel bromodomain and extra-terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma models. BAY 1238097 showed anti-proliferative activity in a large panel of lymphoma-derived cell lines, with a median 50% inhibitory concentration between 70 and 208 nmol/l. The compound showed strong anti-tumour efficacy in vivo as a single agent in two diffuse large B cell lymphoma models. Gene expression profiling showed BAY 1238097 targeted the NFKB/TLR/JAK/STAT signalling pathways, MYC and E2F1-regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling signatures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC-inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented in vitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti-lymphoma preclinical activity in vitro and in vivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK alongside BET bromodomains.

Ruta graveolens extract induces DNA damage pathways and blocks Akt activation to inhibit cancer cell proliferation and survival.

BACKGROUND: Ruta graveolens is a medicinal herb that has been used for centuries against various ailments. This study examined the anticancer properties of the herb using cancer cell lines. MATERIALS AND METHODS: Methanolic extract of R. graveolens was tested on colon, breast and prostate cancer cells. Viability, cell cycle profiles, clonogenicity and capase activation were measured. Induction and subcellular localizations of p53, 53BP1 and γ-H2AX proteins were examined. RESULTS: The extract dose-dependently decreased the viability and the clonogenicity of treated cells and induced G2/M arrest, aberrant mitoses, and caspase-3 activation. It also induced the p53 pathway and focal concentration of the DNA damage response proteins 53BP1 and γ-H2AX. Moreover, the levels of phospho-Akt and cyclin B1 were reduced by treatment, whereas only cyclin B1 was reduced in normal dermal fibroblasts. CONCLUSION: R. graveolens extract contains bioactive compounds which, independently of known photoactivatable mechanisms, potently inhibit cancer cell proliferation and survival through multiple targets.

Effect of hyperbaric oxygen on the anticancer effect of artemisinin on molt-4 human leukemia cells.

BACKGROUND: Artemisinin selectively kills cancer cells which have more intracellular free iron than do normal cells. Hyperbaric oxygen (HBO(2)) may be beneficial in the treatment of cancer. The hypothesis of this study was that HBO(2) enhances anticancer activity of artemisinin. MATERIALS AND METHODS: After pretreatment with 12 µM holotransferrin, Molt-4 human leukemia cells were cultured in 10 µM artemisinin and exposed for 90 min to one of three different conditions: control, room air control, and HBO(2). Cell growth was determined for 48 h after exposure. RESULTS: Differences in growth were noted after 6 h of incubation. After 48 h of incubation, growth of cells treated with artemisinin alone or HBO(2) alone was 85% of that of cells grown under artemisinin-free control conditions. Combined artemisinin and HBO(2) treatment resulted in an additional 22% decrease in growth. CONCLUSION: Combined HBO(2) and artemisinin exposure may be an effective anticancer chemotherapeutic strategy.

Synergistic interaction between platinum-based antitumor agents and demethylcantharidin.

A novel series of TCM-platinum complexes [Pt(C8H8O5)(NH2R)2] 1-5, designed from incorporating demethylcantharidin, a modified component from a traditional Chinese medicine (TCM) with a platinum moiety was found to circumvent cisplatin resistance in mouse leukemia and human hepatocellular carcinoma. These properties are most likely due to the inclusion of the protein phosphatase 2A (PP2A)-inhibiting demethylcantharidin in the novel compounds. We have investigated the potential synergistic effect of combining demethylcantharidin with a platinum-based antitumor agent, such as cisplatin, carboplatin, or oxaliplatin in vitro against L1210 mouse leukemia and SK-Hep-1 human hepatocellular carcinoma, and in vivo against a SK-Hep-1 subcutaneous-inoculated xenograft in nude mice, using median effect analysis. Demethylcantharidin and the platinum antitumor agents were synergistic in all cell lines tested in vitro, and the most effective antiproliferative regimen was when demethylcantharidin was added 24 h before cisplatin. Synergistic antitumor activity was also demonstrated in vivo without undue toxicity; no excessive loss in mouse body weight or overt pathology were observed at the effective doses. The results support a new approach for augmenting cytotoxic effect of established Pt-based drugs with demethylcantharidin in treating human hepatocellular carcinoma and other solid tumors.

Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties.

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analysis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.

Acetone extract of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity.

Bupleuri radix, a traditional Chinese herb, has been widely used to treat liver diseases such as hepatitis and cirrhosis. The acetone extract of Bupleurum scorzonerifolium (AE-BS) showed a dose-dependently antiproliferative effect on the proliferation of A549 human lung cancer cells. The IC(50) of AE-BS, i.e., the concentration required to inhibit proliferation of A549 cells, was 59 +/- 4.5 microg/ml on day 1. The IC(50) of AE-BS for WI38 human normal lung fibroblast cells, however, was significant higher than that for A549 cells (150 +/- 16 microg/ml, p< 0.01). After 72 hours of exposure, AE-BS (60 microg/ml) significantly reduced A549 cell proliferation to 33 +/- 3.2% of control. In TUNEL assay, A549 cells treated with AE-BS showed typical morphologic features of apoptosis, and the percentage of apoptotic cells was approximately 38 % on day 1. In the TRAP assay, AE-BS-treated cells demonstrated significantly lower telomerase activity on day 3. This result indicates that the AE-BS could suppress the proliferation of lung cancer cells via inhibition of telomerase activity and activation of apoptosis.

Green tea polyphenol targets the mitochondria in tumor cells inducing caspase 3-dependent apoptosis.

Induction of apoptosis by green tea polyphenols has been observed in various tumor cell systems, but whether green tea polyphenol-induced apoptosis requires caspase 3 for execution has not been confirmed. We previously reported that green tea polyphenol-induced apoptosis involved Apaf-1 accumulation and caspase 3 activation in the cytosol. In the current study, tumor cells either with deleted caspase 3 gene or expressing wild-type caspase 3 were treated with increasing concentrations of green tea polyphenol(s), followed by morphological analysis and caspase 3 activity assay. The caspase 3 null parental cell line was further examined in comparison with a well-characterized, caspase 3 wild type oral carcinoma cell line by MTT assay and BrdU incorporation assay. The results demonstrated that, while the mitochondrial function gradually declined to insignificant levels, caspase 3 null cells did not undergo apoptosis, which suggested that green tea polyphenol-induced apoptosis is a mitochondria-targeted, caspase 3-executed mechanism.

Aberration in the expression of the retinoid receptor, RXRalpha, in prostate cancer.

There is ample evidence for a role for retinoids in the development and maturation of prostatic epithelium. In recent experiments with conditional disruption of a specific retinoid receptor, namely, RXRalpha in the prostatic epithelium of the mouse, we observed that a major component of retinoid action in the prostate is indeed mediated by RXRalpha. The results clearly indicated that the inactivation of RXRalpha in the prostate epithelium leads to the development of preneoplastic lesions (Huang et al. Cancer Res 62: 4812-9, 2002). To determine the relation of this finding to human prostate cancer, we examined the expression of RXRalpha protein in human prostate cancer cell lines by western blotting and prostate cancer specimens by immunohistochemistry. Relative to the "normal" prostate epithelial cells, there was approximately two- to nine-fold decrease in the full-length 54 kD RXRalpha protein in each of the seven different prostate cancer cell lines tested. Similarly, while RXRalpha immunostaining was uniformly strong in the nuclei of most of the benign prostatic epithelial cells of the thirteen adenocarcinoma specimens tested, a highly heterogeneous pattern of expression was detected in the malignant epithelium, with some areas with low or no staining, others with mostly cytoplasmic staining, and some with both nuclear and cytoplasmic immunoreactivity. To evaluate the effect of RXRalpha modulation on the biologic properties of prostate cancer cell lines, we used a lentivirus expression system to overexpress RXRalpha in CWR22R prostate cancer cells that basally expressed a marginal level of the receptor. The sorted RXRalpha-transduced cells were compared to the corresponding vector control cells for proliferative and apoptotic properties. A correlation of reduction of cell growth or increased susceptibility to apoptosis with increases in the level of RXRalpha nuclear receptor was demonstrated. These effects were further enhanced when the cell culture medium was supplemented with a retinoid receptor panagonist, 9-cis retinoic acid. Together, these data support the notion that, like in mouse prostate, loss or reduction of RXRalpha activity might be a critical factor in prostate tumorigenesis in humans.

Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy.

OBJECT: Ionizing radiation is the gold-standard adjuvant treatment for glioblastoma multiforme (GBM), the most aggressive primary brain tumor. The mechanisms underlying neoplastic glial cell growth inhibition after administration of ionizing radiation, however, remain largely unknown. In this report, the authors characterize the response of GBM cells to ionizing radiation and elucidate factors that correlate with the radiosensitivity of these tumors. METHODS: Six human GBM cell lines were subjected to increasing doses of radiation. Each demonstrated a dose-dependent suppression of cell proliferation. In the most radiosensitive cell line, the authors demonstrated a transient increase in the expression of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27, which corresponded with a G1 cell-cycle arrest. In contrast, the most radioresistant cell line demonstrated a decrease in p21 and p27 expression levels, which correlated with a failure to arrest. Apoptosis did not occur in any cell line following irradiation. Instead, autophagic cell changes were observed following administration of radiation, regardless of the relative radiosensitivity of the cell line. CONCLUSIONS: These findings elucidate some of the molecular responses of GBMs to irradiation and suggest novel targets for future therapy.

[Proapoptotic therapy with oblimersen (bcl-2 antisense oligonucleotide)--review of preclinical and clinical results]. / Proapoptotische Therapie mit Oblimersen (bcl-2-Antisense-Oligonukleotid)--Ubersicht über präklinische und klinische Daten.

The regulation of apoptosis is an important potential target for anticancer therapy. The mitochondrial Bcl-2 protein inhibits apoptosis and is therefore an important mediator of resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy and monoclonal antibody therapy. Oblimersen (Genasense, Aventis Pharmaceuticals / Genta Inc) is a 18mer antisense-oligonucleotide (ASO), which specifically binds to the first 6 codons of the human bcl-2 mRNA, resulting in degradation and destruction of the mRNA by RNAse H. Subsequently there is a significant decrease of bcl-2 translation. A growing number of preclinical and clinical studies suggests that the combination of cytotoxic therapy with Oblimersen results in synergistic anticancer efficacy in many hematologic and solid tumors. Due to its low toxicity profile, oblimersen is an ideal combination partner with conventional chemotherapy. Three randomized phase-III trials (malignant melanoma, chronic lymphocytic leukemia, multiple myeloma) have recently finished recruitment. The results of these studies will be available by the end of 2003. Based on preclinical data, a lot of nonrandomized phase-II studies on several different tumor types like AML, CML, NHL, prostate cancer and breast cancer are underway. The manipulation of proapoptotic and antiapoptotic factors in favor of proapoptotic factors by inhibition of the bcl-2 protein translation in order to enhance the efficacy of anticancer treatments represents a promising new treatment concept in oncology.

UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis.

SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5' regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5' regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

Enhancing the antitumoral effect of hypericin-mediated photodynamic therapy by hyperthermia.

BACKGROUND AND OBJECTIVES: In the previous study, we have found a synergistic effect on the RIF-1 tumor cell killing when hypericin-mediated photodynamic therapy (PDT) was combined with hyperthermia. The purpose of the present study was to investigate the antitumoral effect of hypericin-PDT in combination with hyperthermia in the RIF-1 mouse tumor model. STUDY DESIGN/MATERIALS AND METHODS: Tumor response to PDT in combination with hyperthermia was compared to the response to PDT or hyperthermia alone. To explore the possible mechanism involved in the interaction of PDT and hyperthermia, we determined the tumor cell survival by in vivo/in vitro cell survival assay and analyzed the functional blood vessels by Hoechst 33342 staining. The mode of cell death was examined by TUNEL assay. RESULTS: Enhanced tumor response was obtained by PDT immediately followed by hyperthermia. Tumor cell survival assay revealed that indirect vascular effect contributed greatly to the overall tumor cell death induced by PDT with hypericin, whereas direct tumor cytotoxicity played a major role in hyperthermia-induced tumor cell killing. Combining PDT with hyperthermia brought about a synergistic interaction on direct tumor cell killing. Even though PDT or hyperthermia alone induced severe blood vessel shutdown and the combined treatments led to significant potentiation of the vascular damage as examined by Hoechst staining, the gain in tumor cell death as a result of this secondary vascular effect was limited after the combined treatments. Following the cellular damage by PDT in combination with hyperthermia, tumor cells were triggered to undergo apoptosis. CONCLUSIONS: Our study demonstrated the possibility of using hyperthermia to potentiate the antitumoral effect of hypericin-mediated PDT.

Tetrandrine cytotoxicity and its dual effect on oxidative stress-induced apoptosis through modulating cellular redox states in Neuro 2a mouse neuroblastoma cells.

Tetrandrine (TET), a plant alkaloid, is known primarily as a non-selective Ca(2+) channel blocker. On the contrary to the cytoprotective effect on ischemia/reperfusion injury, TET has also been reported to cause cytotoxicity. In this study, we wished to understand the apparently disparate effects of this potential drug and thus investigated molecular mechanisms on proliferation and apoptosis and its effect on oxidative stress-induced apoptosis in Neuro 2a mouse neuroblastoma cells. We showed that TET, at high concentrations, induced cell cycle arrest and apoptosis through oxidative stress with following observations. Firstly, 10 microM TET elevated the reactive oxygen species (ROS) level and accordingly depleted glutathione (GSH) content. Secondly, pretreatment with antioxidants (NAC or GSH) protected cells from TET-induced apoptosis. We also demonstrated that treatment with 10 microM TET caused not only induction of p53, p21(waf1), and Bax, but also nuclear translocation of p53 and hypo-phosphorylation of pRb concurrently. Our important finding is that the concentration-dependent dual effect of TET, either inhibiting or promoting cell death induced by H(2)O(2) was observed, probably through regulating redox balance, which was well reflected on the GSH content in each condition. Besides, inhibition of Ca(2+) influx protected cells from H(2)O(2)-induced apoptosis even in the presence of 10 microM TET. Taken together, our data suggest that TET regulation of cellular redox states may play a major role in its dual action of cytotoxicity and cytoprotection.

Suppressive effects of tea polyphenol and conformational changes with receptor for advanced glycation end products (RAGE) expression in human hepatoma cells.

BACKGROUND/AIMS: It is reported that polyphenol is associated with low risk of hepatoma and that RAGE (receptor for advanced glycation end products) is important for cancer invasion. METHODOLOGY: Effects of teapolyphenol, EGCG (epigallocatechin-3-gallate) were studied. Proliferation of on human hepatoma cells, HLF, was measured with the use of WST-1 colorimetric assay. Cell invasion was analyzed by the Matrigel invasion assay. Morphology and immunohistological staining of expression of RAGE were also performed. RESULTS: Proliferation was inhibited with the addition of EGCG in a dose-dependent manner. EGCG (200 mumol/L) produced a profound growth suppression of HLF cells (24.5%). Cell invasion was also inhibited with preincubation of 100 mumol/L of EGCG (10.2%). In addition to the antitumor effects, neurite-like conformational changes of HLF cells were observed. Addition of EGCG (100 mumol/L) showed the expression of RAGE on cell surface in accordance to the morphological changes. CONCLUSIONS: The pathway associated to cell movement might be activated with RAGE expression. Although EGCG inhibits the growth and invasion, the cells which expressed RAGE seem to survive. Thus, the enrollment of RAGE should be analyzed to clarify the mechanisms of cancer resistance.

Inhibition of proliferation and induction of apoptosis by tetrandrine in HepG2 cells.

Tetrandrine is a bisbenzylisoquinoline alkaloid derived from the root of Stephania tetrandra S. Moore, which has been reported to elicit in vitro cytotoxic effects on HeLa cells, and in vivo suppressive effects on mouse ascites tumors. In the present study, we examined the antiproliferative and apoptosis-inducing activity of tetrandrine in HepG2 cells, a human hepatoma cell line. Tetrandrine showed potent cytotoxic activity in HepG2 cells (IC(50)=9.0+/-1.0 micro M) following incubation for 48 h. Dose-dependent induction of apoptosis was observed by agarose gel electrophoresis and flow cytometric analysis. Treatment of HepG2 cells with tetrandrine resulted in the activation of caspase-3 protease, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. These results suggest that tetrandrine is potentially useful as a chemotherapeutic/chemopreventive agent in hepatocellular carcinoma.

Anti-angiogenic action of hyperthermia by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro.

Vascular endothelial growth factor is an important angiogenic factor for tumour progression because it increases endothelial-cell proliferation and remodels extracellular matrix in blood vessels. We demonstrated that hyperthermia at 42 degrees C, termed heat shock, suppressed the gene expression and production of vascular endothelial growth factor in human fibrosarcoma HT-1080 cells and inhibited its in vitro angiogenic action on human umbilical vein endothelial cells. The gene expression of alternative splicing variants for vascular endothelial growth factor, VEGF121, VEGF165 and VEGF189, was constitutively detected in HT-1080 cells, but the VEGF189 transcript was less abundant than VEGF121 and VEGF165. When HT-1080 cells were treated with heat shock at 42 degrees C for 4 h and then maintained at 37 degrees C for another 24 h, the gene expression of all vascular endothelial growth factor variants was suppressed. In addition, HT-1080 cells were found to produce abundant VEGF165, but much less VEGF121, both of which were inhibited by heat shock. Furthermore, the level of vascular endothelial growth factor in sera from six cancer patients was significantly diminished 2-3 weeks after completion of whole-body hyperthermia at 42 degrees C (49.9+/-36.5 pg x ml(-1), P<0.01) as compared with that prior to the treatment (177.0+/-77.5 pg x ml(-1)). On the other hand, HT-1080 cell-conditioned medium showed vascular endothelial growth factor-dependent cell proliferative activity and the augmentation of pro-matrix metalloproteinase-1 production in human umbilical vein endothelial cells. The augmentation of endothelial-cell proliferation and pro-matrix metalloproteinase-1 production was poor when human umbilical vein endothelial cells were treated with conditioned medium from heat-shocked HT-1080 cells. These results suggest that hyperthermia acts as an anti-angiogenic strategy by suppressing the expression of tumour-derived vascular endothelial growth factor production and thereby inhibiting endothelial-cell proliferation and extracellular matrix remodelling in blood vessels.

Absence of tumor growth stimulation in a panel of 16 human tumor cell lines by mistletoe extracts in vitro.

Extracts of Viscum album (mistletoe) are widely used as complementary cancer therapies in Europe. The mistletoe lectins have been identified as the main active principle of mistletoe extracts. They have been shown to exhibit cytotoxic effects as well as immunomodulatory activities. The latter is exemplified by induction of cytokine secretion and increased activity of natural killer cells. Recent reports, however, indicated possible tumor growth stimulation by mistletoe extracts. Therefore, the three aqueous mistletoe extracts (Iscador M special, Iscador Qu special and Iscador P) were evaluated for antiproliferative and/or stimulatory effects in a panel of 16 human tumor cell lines in vitro using a cellular proliferation assay. The results show no evidence of stimulation of tumor growth by any of the three Iscador preparations, comprising central nervous system, gastric, non-small cell lung, mammary, prostate, renal and uterine cancer cell lines, as well as cell lines from hematological malignancies and melanomas. On the contrary, Iscador preparations containing a high lectin concentration (Iscador M special and Iscador Qu special) showed antitumor activity in the mammary cancer cell line MAXF 401NL at the 15 microg/ml dose level with a more than 70% growth inhibition compared to untreated control cells. In addition, a slight antitumor activity (growth inhibition 30-70%) was found in three tumor cell lines for Iscador M special and in seven tumor cell lines for Iscador Qu special, respectively. Iscador P, which contains no mistletoe lectin I, showed no antiproliferative activity.

Blocking HSF1 by dominant-negative mutant to sensitize tumor cells to hyperthermia.

Heat shock protein 70 (HSP70), an antiapoptotic chaperon protein, is highly expressed in human breast tumors and renders them resistant to such therapy as hyperthermia. In the present study, we inhibited the expression of HSP70 by blocking the heat shock transcription factor 1 (HSF1) function with its dominant-negative mutant (mHSF1) in Bcap37 cells, a thermotolerant breast cancer cell line. Here we report that retrovirus-mediated transfer of mHSF1 led to massive cell death of Bcap37 after hyperthermia. mHSF1 sensitized Bcap37 cells to hyperthermia by promoting apoptosis induced by heat shock. We also examined the efficacy of mHSF1 gene therapy in the nude mouse. mHSF1 transfection led to diminution of tumor growth with hyperthermia therapy. Thus, disrupting HSF1 in combination with hyperthermia may open new possibilities for treatment of cancers that have acquired resistance to heat treatment.