Taiwanin A induced cell cycle arrest and p53-dependent apoptosis in human hepatocellular carcinoma HepG2 cells

Taiwanin A, a lignan isolated from Taiwania cryptomerioides Hayata, has previously been reported to have cytotoxicity against human tumor cells, but the mechanisms are unclear. In this study, we examined the molecular mechanism of cell death of human hepatocellular carcinoma HepG2 cells induced by Taiwanin A. Taiwanin A has been found to induce cell cycle arrest at G2/M phase as well as caspase-3-dependent apoptosis within 24 h. We performed both in vitro turbidity assay and immunofluorescence staining of tubulin to show that Taiwanin A can inhibit microtubule assembly. Moreover, the tumor suppressor protein p53 in HepG2 cells was activated by Taiwanin A within 12 h. Inhibition of p53 by either pifithrin-alpha or by short hairpin RNA which blocks p53 expression attenuates Taiwanin A cytotoxicity. Our results demonstrate that Taiwanin A can act as a new class of microtubule damaging agent, arresting cell cycle progression at mitotic phase and inducing apoptosis through the activation of p53.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Interferon-gamma enhances target cell sensitivity to monocyte killing

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.     

Cytotoxic activity of halogenated monoterpenes from Plocamium cartilagineum

Nine halogenated monoterpenes isolated from the red alga Plocamium cartilagineum have been evaluated for their cytotoxic effects on the tumor cell lines CT26 (murine colon adenocarcinoma), SW480 (human colon adenocarcinoma), HeLa (human cervical adenocarcinoma) and SkMel28 (human malignant melanoma) with several multidrug resistance mechanisms and the mammalian non-tumor cell line CHO (Chinese hamster ovary cells). The activities of these compounds were compared with those of the insecticide gamma-hexachlorocyclohexane (lindane) due to chemical structure similarities. Compounds 1, 2, 3, and 5 exhibited selective cytotoxicity against colon and cervical adenocarcinoma cells. Interestingly, the effect of compound 3 was specific and irreversible to human colon adenocarcinoma SW480 cells, which overexpress the transmembrane P-glycoprotein often related to chemoresistance. None of the anti-tumor doses of these compounds was cytotoxic against CHO cells. Furthermore, analysis of cellular extracts after incubation with the test compounds and rotenone (positive uptake control) demonstrated the intracellular accumulation of 1, 2, 3, and 5.     

In vitro cytotoxicity study on platinum (II) complexes with epoxysuccinates as leaving groups

A series of novel cisplatin-type platinum complexes were designed, characteristic of epoxysuccinates as leaving groups. The pertinent compounds were prepared and characterized by IR, (1)H NMR, and ESI-MS spectra with elementary analyses. The in vitro cytotoxic activities of compounds toward SPC-A1 human lung adenocarcinoma cell line and BGC823 human stomach adenocarcinoma cell line were determined. Biological tests have confirmed that complexes containing 4R,5R-DMID [abbreviation of (4R,5R)-4,5-bis (aminomethyl)-2-isopropyl-1,3-dioxolane] as carrier ligands have greater cytotoxicity toward tumor cells than the corresponding compounds with other carrier ligands. Most platinum complexes with trans-epoxysuccinates usually have higher cytotoxicity than those with cis-epoxysuccinates. Complex 4a shows the most effective among those tested platinum complexes in both cell lines, and its cytotoxicity approached that of cisplatin.     

Do conjugated eicosapentaenoic acid and conjugated docosahexaenoic acid induce apoptosis via lipid peroxidation in cultured human tumor cells?

Conjugated eicosapentaenoic acid (CEPA) and conjugated docosahexaenoic acid (CDHA) with triene structure, isomerized by alkaline treatment, showed intensive cytotoxicity with LD(50) at 12 and 16 microM, respectively, in DLD-1 cells (colorectal adenocarcinoma), while they had no effect on normal human fibroblast cell lines such as MRC-5, TIG-103, and KMS-6 cells. Cytotoxic action of CEPA and CDHA was also demonstrated in other tumor cell lines including HepG2, A549, MCF-7, and MKN-7 cells. alpha-Tocopherol suppressed cytotoxicity of CEPA and CDHA in tumor cells, and the cytotoxicity involved membrane phospholipid peroxidation. CEPA and CDHA induced DNA condensation and fragmentation in DLD-1 cells, indicating the involvement of apoptosis in this cytotoxic mechanism. Furthermore, previous reports have shown that lipid peroxidation product induces cell death, including apoptotic cell death in different cell lines. CEPA and CDHA have been demonstrated in cultured cells to cause cell death via lipid peroxidation and apoptosis in the absence of alpha-tocopherol.     

Cytostatic and growth-stimulating effects of alveolar macrophages of rats on tumor cells

Cytostatic and growth-stimulating effects of alveolar macrophages (AM) of rats on tumor cells were studied. The experimental results are summarized as follows. 1. The cytotoxicity of AM activated with BCG to tumor cells was increasing with the increase of effector cells/target cells (E/T) ratio. AM without the treatment with BCG expressed slight cytotoxicity to tumor cells at a high E/T, and growth-stimulating effect on tumor cells, at a low E/T. 2. AM after 24-hour culture had a lower manifestation of cytotoxicity to human lung adenocarcinoma cell line than that of AM without 24-hour culture, and had a growth-stimulating effect on B-16 cell line. 3. Cytostatic and growth-stimulating effects of AM without or with 24-hour culture were decreasing with the increase of irradiation doses.     

裸鼠肿瘤动物模型VEGF受体表达及其意义

目的　通过免疫组织化学染色了解flt 1与flk 1 KDR(VEGF的两个高亲和受体 )在人肿瘤细胞皮下接种肿瘤动物模型的血管内皮细胞与肿瘤细胞中的表达。方法　取荷瘤裸鼠皮下接种瘤块 ,漂洗、固定、石蜡连续切片 ,进行两种受体相应免疫组化检测。结果　在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中flt 1的阳性率大部分为强阳性或中阳性 ,而只有在荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中flt 1的阳性率为弱阳性 ,在荷人卵巢癌SKOv3裸鼠的肿瘤细胞中flt 1的表达为阴性。相比较而言 ,在 13种荷瘤裸鼠血管内皮细胞及肿瘤细胞中KDR的阳性率大部分为中阳性或弱阳性 ,并且在荷人肝癌SMMC 772 1裸鼠 ,荷人胃腺癌SPC A1裸鼠 ,荷人高转移肝癌移植瘤裸鼠 ,荷人卵巢癌SKOv3裸鼠的肿瘤细胞中 ,荷人宫颈癌移植瘤裸鼠和荷人胃腺癌MKN 4 5裸鼠的肿瘤细胞中 ,KDR表达为阴性。结论　VEGF受体共同表达于肿瘤血管内皮细胞与肿瘤细胞 ,提示了VEGF与VEGF受体结合作用在肿瘤演化中的重要性 ,为靶向于VEGF受体的基因治疗策略选择裸鼠动物模型提供了参考依据     

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.     

Induction of activated natural killer cells from murine spleen cells primed in vivo and subsequently challenged in vitro with the streptococcal preparation OK432

Summary The present study shows that natural killer cell-mediated cytotoxicity of BALB/c mouse spleen cells to syngeneic tumor cells was augmented by in vivo priming or in vitro stimulation with the streptococcal preparation OK432. The augmentation of spleen cell cytotoxicity to syngeneic tumor cells by in vivo priming alone with OK432 was lower than that obtained by in vitro stimulation alone with OK432. When the murine spleen cells primed in vivo with OK432 were rechallenged in vitro with OK432 at various intervals, the natural cytotoxicity was more strongly enhanced than that seen with in vitro stimulation alone. The cell surface phenotype of killer cells activated with OK432 was Thy 1⁺ and asialo GM _inf1 ^sup+ , suggesting the activated natural killer cell. Next, mice were transplanted with syngeneic colon adenocarcinoma cells, and primed in vivo with OK432. These spleen cells were subsequently challenged in vitro with OK432. These spleen cells displayed a strong cytotoxic activity not only to the transplanted adenocarcinoma cells but also to other syngeneic tumor cells.     

Antitumor activity of L-asparaginase from Erwinia carotovora against different human and animal leukemic and solid tumor cell lines

The dose- and time-dependent antitumor and cytotoxic effects of L-asparaginases from Erwinia carotovora (ECAR LANS) and Escherichia coli (MEDAC) have been investigated using human leukemic cells and human and animal solid tumor cells. These included human T-cell acute lymphoblastic leukemia cell lines (Jurkat, Jurkat/A4, Molt-4), human chronic myeloid leukemia K562 cells, human promyelocytic leukemia HL-60, and also human solid tumor cells (prostate carcinoma LnCap, breast adenocarcinoma MCF7, ovarian adenocarcinoma SCOV-3 and carcinoma CaOV, hepatocarcinoma Hep G2, fibrosarcoma HT-1080) and animal solid tumor cells (rat Gasser??s ganglion neurinoma cells GGNC-1, mouse glioblastoma EPNT-5). We investigated sensitivity of tumor cells (seeded at different density) to L-asparaginases, as well the effect of L-asparaginases on cell growth rate, protein and DNA synthesis in the presence of various cytostatics. Cell cycle analysis by flow cytofluorimetry and detection of apoptotic cells before and after treatment with L-asparaginases indicate that ECAR LANS L-asparaginase suppressed growth of all tested solid tumor cells. Evaluation of leukemic cell number after treatment with L-asparaginases for 24, 48 and 72 h demonstrated that asparagine deficiency did not kill cells but stopped normal cell division. The cytofluorometric study of solid and leukemic cells revealed that except HL-60 cells the treatment with L-asparaginase for 72 h did not change cell cycle phase distribution and did not increase the number of apoptotic cells. Combined treatment of cells using a combination of L-asparaginase and doxorubicin significantly increased the number of apoptotic cells up to 60% (MCF-7 cells), 40% (Jurkat cells) and even 99% (HL-60). High levels of DNA and protein synthesis rates in asparaginase-treated tumor cells suggest lack of massive entry of tumor cells to apoptosis. This conclusion is based on the fact of sensitivity of multi-resistant Jurkat/A4 cells to L-asparaginases (it is nearly impossible to induce apoptosis in these cells). Since ECAR LANS did not influence growth of normal human fibroblasts it appears that the enzyme cytotoxicity is associated only asparagine deficiency.     

NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors

NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.     

Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.

To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.     

Cytotoxic action of natural killer cells on human tumor cells

Natural killer cell cytotoxicity was studied in a 18-hour 51Cr-release assay in the cultures of human tumor target cells: K562 leukemia and lung adenocarcinoma (LAC) cells. The mean cytotoxic value was similar for K562 and LAC cells: 36.13 +/- 3.23% and 40.78 +/- 3.43%, respectively, although significant individual variability was recorded. The similar cytolytic action of blood mononuclear cells (MNC) on the two tumor lines was observed in 30% of normal donors. MNC from 30% donors produced more pronounced lytic action on K562 cells while MNC from other 30% donors lysed mainly LAC cells. In the competitive inhibition test cold K562 cells more effectively than cold LAC cells suppressed the MNC-induced lysis of both K562 and LAC cells.     

Assessment of the activity of tumor necrosis factor in normal state and in ovarian tumors

The cytotoxic activity of tumor necrosis factor of healthy individuals and patients with cystadenoma and adenocarcinoma of ovaries was investigated. Tumor necrosis factor (TNF) spontaneous activities of healthy donors and patients with cystadenoma of ovaries was the same. The level of TNF spontaneous activity of patients with adenocarcinoma was increased. LPS-induced TNF cytotoxicity in patients with cystadenoma was decreased. Mononuclear cells of patients with adenocarcinoma did not respond to LPS stimulation by increased TNF production.     

Design,Synthesis, and In Vitro and In Vivo Biological Studies of a 3′-Deoxythymidine Conjugate that Potentially Kills Cancer Cells Selectively

Thymidine kinases (TKs) have been considered one of the potential targets for anticancer therapeutic because of their elevated expressions in cancer cells. However, nucleobase analogs targeting TKs have shown poor selective cytotoxicity in cancer cells despite effective antiviral activity. 3′-Deoxythymidine phenylquinoxaline conjugate (dT-QX) was designed as a novel nucleobase analog to target TKs in cancer cells and block cell replication via conjugated DNA intercalating quinoxaline moiety. In vitro cell screening showed that dT-QX selectively kills a variety of cancer cells including liver carcinoma, breast adenocarcinoma and brain glioma cells; whereas it had a low cytotoxicity in normal cells such as normal human liver cells. The anticancer activity of dT-QX was attributed to its selective inhibition of DNA synthesis resulting in extensive mitochondrial superoxide stress in cancer cells. We demonstrate that covalent linkage with 3′-deoxythymidine uniquely directed cytotoxic phenylquinoxaline moiety more toward cancer cells than normal cells. Preliminary mouse study with subcutaneous liver tumor model showed that dT-QX effectively inhibited the growth of tumors. dT-QX is the first molecule of its kind with highly amendable constituents that exhibits this selective cytotoxicity in cancer cells.     

miR-146b Reverses epithelial-mesenchymal transition via targeting PTP1B in cisplatin-resistance human lung adenocarcinoma cells

Epithelial-mesenchymal transformation (EMT) is associated with drug resistance in human lung adenocarcinoma cells, but its specific mechanism has not been clarified. In this study, we investigated the effect of miRNA-146b on EMT in cisplatin (DDP) resistant human lung adenocarcinoma cells and the corresponding mechanism. Cisplatin resistant (CR) human lung adenocarcinoma cells (A549/DDP and H1299/DDP) were established, and the EMT characteristics and invasion and metastasis ability of CR cells were determined by tumor cell-related biological behavior experiments. The role of miR-146b in EMT of CR cells was determined by in vitro functional test. The targeted binding of miR-146b to protein tyrosine phosphatase 1B (PTP1B) was verified by biological information and double luciferin gene reporting experiments. The effect of miR-146b on tumor growth and EMT phenotype in vivo was investigated by establishing the xenotransplantation mouse model. Compared with the control group, H1299/DDP and A549/DDP cells showed the enhanced EMT phenotypes, invasion and migration ability. Besides, miR-146b was lowly expressed in H1299/DDP and A549/DDP cells. More importantly, overexpressed miR-146b could specifically bind to PTP1B, thus inhibiting the EMT process and ultimately reducing CR in H1299/DDP and A549/DDP cells. Finally, overexpressed miR-146b observably inhibited tumor growth in xenograft model mice and inhibited the EMT phenotype of A549/DDP cells in vivo by regulating the expressions of EMT-related proteins. Overexpressed miR-146b could reverse the EMT phenotype of CR lung adenocarcinoma cells by targeting PTP1B, providing new therapeutic directions for CR of lung adenocarcinoma cells.     

A fibroblast-derived tumor cytotoxic factor/F-TCF (hepatocyte growth factor/HGF) has multiple functions in vitro.

We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth factors (hHGFs). In the present report, we demonstrate its biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxicity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, HUVEC and human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, HL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver.     

Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis.

The endoglycosidase heparanase is an important in the degradation of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Here we report the cDNA sequence of the human platelet enzyme, which encodes a unique protein of 543 amino acids, and the identification of highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlates with their metastatic potential. Exhaustive studies have shown only one heparanase sequence, consistent with the idea that this enzyme is the dominant endoglucuronidase in mammalian tissues.     

A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer

Background

Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).

Results

The cc49scFv-FasL_ext is highly effective in in vitro killing of human TAG-72⁺ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasL_ext only increased cytotoxicity 500-fold as compared with sFasL against TAL6⁺ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasL_ext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.

Conclusion

Our study demonstrated that scFv-FasL_ext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.