5k, a novel ß-O-demethyl-epipodophyllotoxin analogue, inhibits the proliferation of cancer cells in vitro and in vivo via the induction of G2 arrest and apoptosis.

Etoposide (VP-16), a topoisomerase II (Topo II) inhibitor, has been widely used to treat malignancies. Its clinical application, however, has been hindered by the rise of acquired multidrug resistance (MDR). Here, we report that 4ß-{[4-(pyrrolidin-1-ylmethyl)phenyl]amino}-4'-O-Demethyl-4-Epipodophyllotoxin (5k), a novel ß-O-demethyl-epipodophyllotoxin analogue, possesses higher antitumor activity than its parent compound (VP-16) in a panel of various human tumor cell lines. More importantly, it was also effective against MDR cells both in vitro and in vivo. Using a KB/VCR MDR tumor xenograft model that overexpresses P-gp, 5k (2.5 mg/kg) exhibited a 2.4-fold higher growth inhibition rate versus VP-16 (5 mg/kg). In contrast, 5k and VP-16 displayed similar antitumor activities in a KB tumor xenograft model. Molecular and cellular mechanism studies revealed that 5k targeted Topo II by trapping DNA-Topo II cleavage complexes that could directly cause DNA damage. There were two distinct cellular responses to DNA damage elicited by the treatment with 5k: at low concentrations (20-80 nM), mitotic entry was arrested through the suppression of the activity of Cyclin B1/Cdc 2 complexes via the ATM/ATR signaling pathway; at high concentrations (1.25-5.00 µM), 5k-induced apoptotic signaling was mediated by the mitochondrial death pathways. Collectively, these data demonstrate the potential value of 5k as an antitumor drug candidate that should be further developed.

Calpain-mediated androgen receptor breakdown in apoptotic prostate cancer cells.

Since androgen receptor (AR) plays an important role in prostate cancer development and progression, androgen-ablation has been the frontline therapy for treatment of advanced prostate cancer even though it is rarely curative. A curative strategy should involve functional and structural elimination of AR from prostate cancer cells. We have previously reported that apoptosis induced by medicinal proteasome-inhibitory compound celastrol is associated with a decrease in AR protein levels. However celastrol-stimulated events contributing to this AR decrease have not been elucidated. Here, we report that a variety of chemotherapeutic agents, including proteasome inhibitors, a topoisomerase inhibitor, DNA-damaging agents and docetaxel that cause cell death, decrease AR levels in LNCaP prostate cancer cells. This decrease in AR protein levels was not due to the suppression of AR mRNA expression in these cells. We observed that a proteolytic activity residing in cytosol of prostate cancer cells is responsible for AR breakdown and that this proteolytic activity was stimulated upon induction of apoptosis. Interestingly, proteasome inhibitor celastrol- and chemotherapeutic drug VP-16-stimulated AR breakdown was attenuated by calpain inhibitors calpastatin and N-acetyl-L-leucyl-L-leucyl-L-methioninal. Furthermore, AR proteolytic activity pulled down by calmodulin-agarose beads from celastrol-treated PC-3 cells showed immunoreactivity to a calpain antibody. Taken together, these results demonstrate calpain involvement in proteasome inhibitor-induced AR breakdown, and suggest that AR degradation is intrinsic to the induction of apoptosis in prostate cancer cells.

Cyclosporin A inhibits HTLV-I tax expression and shows anti-tumor effects in combination with VP-16.

Adult T cell leukemia (ATL) is one of the most refractory malignant hematological diseases. Our previous studies demonstrated HTLV-1Tax protein involvement in clinical manifestation of the aggressive type of ATL and suggested the potential application of agents to inhibit Tax expression for ATL treatment. In the present study, we first examined Tax involvement in the resistance to VP-16-induced apoptosis using four HTLV-1 infected T cell clones and cTax DNA-transfected cells. Next, we examined whether cyclosporin A reduced expression of Tax and its related transfer factors on Western blot and CAT assay. We further investigated whether cyclosporin A in combination with VP-16 can induce apoptosis in HTLV-1 infected T cells. Tax-producing T cells, K3T and F6T, were resistant to VP-16 induced growth inhibition compared with that of the nonproducing cells, S1T and Su9T01. Experiments using S1T and Tax-expressing cDNA-transfected S1T demonstrated Tax-induced resistance to VP-16 induction of apoptosis by DNA ladder formation. Cyclosporin A reduced Tax expression in K3T by Western blot analysis and on CAT assay, showing maximal reduction of 61% and 60% compared to control culture using LTR CAT transfected Jurkat cells and K3T cells, respectively. Cyclosporin A also reduced the nuclear expression of two Tax-related transfer factors, ATF-1 and ATF-2 on Western blot. Cyclosporin A alone did not show any cytotoxicity by itself, but sensitized cells to VP-16 when combined with VP-16. Cyclosporin A may be a useful anti-ATL agent when combined with other anti-cancer agents possibly related to Tax inhibition.

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference.

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.

Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16.

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.

Topologically constrained domains of supercoiled DNA in eukaryotic cells.

The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51+/-0.17Mbp and 1.34+/-0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.

Atypical apoptotic cell death induced in L929 targets by exposure to tumor necrosis factor.

The mechanism by which tumor necrosis factor (TNF) induces cytotoxicity of murine fibroblasts was investigated. Electrophoresis of DNA extracted from TNF-treated L929 targets showed fragmentation of DNA into a ladder-like pattern, typical of cells dying by apoptosis. Morphologic analysis also indicated apoptotic cell death, demonstrating clumping and crescentic condensation of chromatin. In contrast, chromatin condensation and ladder-like DNA fragmentation were not detected in L929 targets dying by necrosis from exposure to heat, repeated cycles of freeze-thaw, and sodium azide. Chromatin condensation was an early event, detected as early as 6 h of incubation. However, DNA fragmentation (assayed by double-stranded fragmentation assay and gel electrophoresis), as well as the apoptotic changes detected by Hoechst fluorescence, both occurred later and did not precede TNF cytotoxicity (membrane permeabilization detected by trypan blue or propidium iodide staining). This atypical pattern of apoptosis was a characteristic of L929 target cells rather than a generalized cytotoxic response to TNF because TNF-treated squamous cancer cells showed typical features of apoptosis (DNA fragmentation before cytotoxicity) and etoposide-treated L929 cells demonstrated the same atypical kinetics as TNF-treated cells. Zinc significantly inhibited TNF cytotoxicity as well as DNA fragmentation of L929. However, because DNA fragmentation occurred belatedly in TNF-treated targets, lagging behind cytotoxicity, the protection by zinc against TNF appears mediated by events that occur before the ultimate endonuclease-induced cleavage of DNA into small fragments.

Recombinant activation domains of virion protein 16 and human estrogen receptor generate transcriptional interference in vitro by distinct mechanisms.

Overexpression of transcriptional activators in transfection assays may inhibit their own activity or interfere with trans-activation by different sequence-specific transcription factors. In this study we show that this phenomenon of transcriptional interference (squelching) can be mimicked in vitro by adding recombinant activation domains to nuclear extracts. We demonstrate that the acidic activation domain of virion protein 16 interferes both with basal transcription from TATA-box promoters and promoters activated by various trans-activators in two different mammalian cell-free transcription systems. This suggests that virion protein 16 interacts with and thereby sequesters a basal transcription factor. In contrast the recombinant activation function 2 (AF-2) of human estrogen receptor does not affect basal promoter activity but inhibits TATA promoters activated by human progesterone receptor (hPR) or Sp 1 as well as the beta-globin and adenovirus major late promoter. By analyzing the effects of AF-2 on DNA binding of hPR and Sp1 we found that AF-2 inhibits the DNA binding activity of hPR, but not Sp1. Our data suggest that the recombinant AF-2 squelches Sp1 trans-activation by sequestering a common coactivator(s), whereas hPR function might be inhibited due to competition for a common cofactor stabilizing hPR dimers or through the formation of inactive heterodimers between AF-2 and hPR.

Reduced DNA topoisomerase II in VP-16 resistant mouse breast cancer cell line.

VP-16 resistant cells, FvprB350 (50B-3), were isolated from mouse breast cancer cell line FM3A. 50B-3 cells showed 84-fold higher resistance than their parent cells. Reduced drug uptake was not found in resistant cells. Quantitative analysis of drug-stimulated DNA cleavage activity using 3'32P end-labeled pBR322 restriction fragments showed that VP-16 stimulated DNA-topoisomerase II cleavable complex forming activity in crude nuclear extract from 50B-3 cells was approximately one-fifth as compared with that of FM3A wild-type cells. Dot-blot analysis of RNA extracted from the two cell lines showed that mRNA levels of topoisomerase II in 50B-3 cells drastically decreased and catalytic activity was also 1/2-1/3 as compared with that of parent cells. 50B-3 cells showed cross resistance to VM-26, m-AMSA, adriamycin. These findings suggest that reduced topoisomerase II activity (cellular levels) and cleavable complex forming activity may be significant factors in the marked drug resistance.