K.N. Leung

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 ?g/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.

Induction of tumor necrosis factor receptor type 2 gene expression by tumor necrosis factor-α in rat primary astrocytes

Using reverse transcription-polymerase chain reaction (RT-PCR) technique, the messenger RNA (mRNA) for tumor necrosis factor receptor type 2 (TNF-R2, 75/80 kDa) was detected in rat primary astrocytes, with much lower level of expression when compared to that for tumor necrosis factor receptor type 1 (TNF-R1, 55/60 kDa). Upon exposure to TNF-alpha (100 U/ml), the TNF-R2 mRNA level was greatly enhanced at 8 h, while TNF-R1 mRNA remained unchanged even after 24 h. The induction of TNF-R2 gene expression by TNF-alpha was dose-dependent and seemed to be unique to TNF-alpha, as interleukin-6 (IL-6) had no significant effect on TNF-R2 expression. Since TNF-R2 was reported to mediate mitogenic and gene-inducing effects in many other cell types, it is likely that the reported proliferative effect of TNF-alpha on astrocytes was also mediated by this TNF receptor subtype. Upon exposure to TNF-alpha or lipopolysaccharide (LPS), the expression of TNF-alpha gene was induced, and the LPS-induced TNF-alpha seemed to selectively enhance the TNF-R2 gene expression. Collectively, our results suggest that the TNF-alpha or LPS-induced expression of both TNF-R2 and TNF-alpha may provide a positive control mechanism to further enhance the proliferative effect of TNF-alpha in astrocytes.

Tryptanthrin Inhibits Angiogenesis by Targeting the VEGFR2-Mediated ERK1/2 Signalling Pathway

Angiogenesis is a key step for tumour growth and metastasis, and anti-angiogenesis has been proposed as an important strategy for cancer therapy. Tryptanthrin is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants and has been shown to possess anti-tumour activities on various cancer cell types. This study aims to investigate the in vitro and in vivo anti-angiogenic activities of tryptanthrin and to unravel its underlying molecular action mechanisms. Our results show that tryptanthrin inhibited the in vitro proliferation, migration, and tube formation of the human microvascular endothelial cells (HMEC-1) in a concentration-dependent manner and significantly suppressed angiogenesis in Matrigel plugs in mice. Mechanistic studies indicated that tryptanthrin reduced the expression of several pro-angiogenic factors (Ang-1, PDGFB and MMP2). Tryptanthrin was also found to suppress the VEGFR2-mediated ERK1/2 signalling pathway in HMEC-1 cells and molecular docking simulation indicated that tryptanthrin could bound to the ATP-binding site of VEGFR2. Collectively, the present study demonstrated that tryptanthrin exhibited both in vitro and in vivo anti-angiogenic activities by targeting the VEGFR2-mediated ERK1/2 signalling pathway and might have therapeutic potential for the treatment of angiogenesis-related diseases.

Activation of the anti-tumor effector cells by Radix bupleuri

Radix bupleuri, the root of Bupleuri spp., Chinese medicinal herbs used for the treatment of influenza, malaria and menstrual disorders, were extracted with hot water and separated into five different fractions (RB, RBI, RBII, RBIII and RBIV) by stepwise alcohol precipitation. One of these fractions, RBI, was then fractionated into RBIa and RBIb by gel filtration using G-100 Sephadex. These two fractions were further purified into RBIai, RBIaii and RBIbi, RBIbii fractions respectively by ion-exchange chromatography using DEAE-Sephadex. Each of these fractions is a heteropolymer consisting mainly of carbohydrate and varying proportions of protein and uronic acid. RBIaii was found to show strong anti-tumor activities in sarcoma-bearing mice. Mechanistic studies showed that RBIaii exhibited a potent activating effect on the cytotoxic activity of macrophages, NK and LAK cells against tumor cells. In addition, RBIaii could increase the number of tumor infiltrating lymphocytes (TILs) in the tumor site of WEHI-164-bearing mice. Furthermore, RBIaii could induce the release of interferon-gamma by lymphocytes in vitro.

Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells.

ETHNOPHARMACOLOGICAL RELEVANCE Agaricus blazei has been used as an adjuvant in cancer chemotherapy and is found to inhibit the growth of various types of tumor cells. AIM OF THE STUDY Our study has adopted a systematic and bioassay-guided approach to optimize the extraction of Agaricus blazei for anti-leukemic bioactive components. The tumor-selective growth inhibitory activity of the extracts on leukemic cell lines was evaluated in vitro and in vivo using tumor-bearing nude mice. MATERIALS AND METHODS Agaricus blazei extracts were prepared using different methods. MTT and tritiated thymidine incorporation assays were used to evaluate the in vitro anti-leukemic effects. The most potent extract was further investigated using NB-4 cells-bearing nude mice and mechanistic studies using DNA fragmentation assay and cell death detection ELISA. RESULTS The JAB80E70 extract showed the most potent tumor-selective growth inhibitory activity against human leukemia NB-4 and K-562 cells. This is the first report of anti-leukemic activity of JAB80E70 in athymic nude mice bearing NB-4 cells. Using DNA fragmentation assays and cell death detection ELISA, JAB80E70 was found to induce apoptosis in NB-4 cells. However, the polysaccharide enriched fractions failed to show significant cytotoxicity on NB-4 cells in vitro. CONCLUSIONS The JAB80E70 extract exhibited potent anti-leukemic effect in vitro and in vivo. The effect can be attributed, at least in part, to the induction of apoptosis. Besides, polysaccharides in Agaricus blazei may not possess direct anti-leukemic activity in vitro.

Tumor necrosis factor-α mediates the proliferation of rat C6 glioma cells via β-adrenergic receptors

In the present study, we observed that isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, stimulated rat C6 glioma cell proliferation, while propranolol, a beta-AR blocker, greatly reduced the proliferative effect of TNF-alpha on C6 cells. The gene and protein expressions of both beta1- and beta2-ARs were enhanced in C6 cells after TNF-alpha treatment, and the increase in beta-AR was due to an increased number of binding sites and not due to increase in receptor affinity. We further showed that protein kinase C (PKC) was involved in the TNF-alpha-induced beta-AR expression. Collectively, our results indicate that TNF-alpha-induced proliferation in C6 glioma cells might be via the induction and activation of beta-ARs.

Cytokines in the Differentiation Therapy of Leukemia: From Laboratory Investigations to Clinical Applications

Differentiation therapy of leukemia is the treatment of leukemia cells with biological or chemical agents that induce the terminal differentiation of the cancer cells. It is regarded as a novel and targeted approach to leukemia treatment, based on our better understanding of the hematopoietic process and the mechanisms of its deregulation during leukemogenesis. Clinically, differentiation therapy has been most successful in acute promyelocytic leukemia using all-trans-retinoic acid as the inducer, either alone or in combination with chemotherapy. This review presents evidence that a number of hematopoietic cytokines play important roles in both normal and aberrant hematopoietic processes. In vitro laboratory investigations in the past two decades using well-characterized myeloid leukemic cell lines and primary blast cells from leukemia patients have revealed that many hematopoietic cytokines can trigger lineage-specific differentiation of leukemia cells, which may have important implications in the clinical setting. Moreover, our current understanding of cytokine interactions and the molecular mechanisms of cytokine-induced leukemic cell differentiation will be discussed in the light of recent findings. Finally, ways in which laboratory research on cytokines in the differentiation therapy of leukemia can lead to the improved design of protocols for future clinical applications to leukemia therapy will also be addressed.

Selective induction of tumor necrosis factor receptor type II gene expression by tumor necrosis factor-α in C6 glioma cells

Using reverse transcription-polymerase chain reaction (RT-PCR) technique, the levels of tumor necrosis factor receptors gene expression in C6 glioma cells upon induction with tumor necrosis factor-alpha (TNF-alpha) were analysed. In control cells, the level of mRNA for tumor necrosis factor receptor type II (TNF-R2; 75/80 kDa) was much lower than that of tumor necrosis factor receptor type I (TNF-R1; 55/60 kDa). Upon exposure to TNF-alpha, the TNF-R2 mRNA level was greatly increased, while the TNF-R1 mRNA level remained unchanged even after 48 h. The induction of TNF-R2 gene expression by TNF-alpha was dose-dependent and seemed to be unique to TNF-alpha, as IL-6 had no effect. Since TNF-R2 was reported to mediate mitogenic effect in many other cell types, it is likely that the reported proliferative effect of TNF-alpha on astrocytes and C6 glioma cells was mediated by this TNF receptor subtype.

Involvement of tumor necrosis factor (TNF-α) in arsenic trioxide induced apoptotic cell death of murine myeloid leukemia cells

Arsenic trioxide (As(2)O(3)) has recently been shown to be effective to inhibit the growth and to induce apoptosis in acute promyelocytic leukemia (APL) but not in acute myeloid leukemia (AML) cells. Recently, we have isolated an As(2)O(3) sensitive subclone JCS-16 from the murine myeloid leukemia WEHI 3B (JCS). At the concentrations of 0.3-3 microM, As(2)O(3) induces a dose-dependent cytotoxicity and growth inhibition on the JCS-16 cells. As(2)O(3) also induces apoptotic cell death, as judged by the presence of apoptotic nuclei, at 6 h after treatment. Morphological differentiation was not observed in As(2)O(3) treated JCS cells. Neutralizing anti-TNF-alpha antibody was found to reduce the As(2)O(3)-mediated apoptotic cell death of JCS-16 cells. Growth inhibitory effect of As(2)O(3) was also reduced after the addition of anti-TNF-alpha. In addition, reverse transcription polymerase chain reaction (RT-PCR) and reverse northern blot analysis demonstrated that the expression of TNF receptor (TNF-R2), IL-4, and IL-4R was down-regulate at 1 h after As(2)O(3) treatment. The expression of TNF-alpha and TNF-R1 was not affected. Our results suggest that the autocrine action of TNF-alpha might play a role in As(2)O(3)-induced apoptotic cell death of JCS-16 leukemia cells.

EFFECTS OF BIOCHANIN A ON THE GROWTH AND DIFFERENTIATION OF MYELOID LEUKEMIA WEHI-3B (JCS) CELLS

The effects of biochanin A on the growth and differentiation of a recently characterized myeloid leukemia cell line WEHI-3B (JCS) were investigated. Biochanin A not only inhibited the growth of JCS cells in a dose-dependent manner (0 - 200 microM) but also induced the morphological differentiation of JCS cells. The phagocytic activity of biochanin A-treated JCS cells was also increased. Flow cytometric analysis showed that the expression of macrophage differentiation markers Mac-1 and F4/80 was up-regulated in biochanin A-treated JCS cells. The expression level of Mac-1 was higher than that of F4/80. The expression of cytokine genes was studied by reverse transcription-polymerase chain reaction (RT-PCR) and cycle titration. mRNA levels of IL-1alpha, IL-1beta and IL-4 were found to be up-regulated at 46 hours after incubation of JCS cells with biochanin A. Although the expression of LIF was also up-regulated, the LIF receptor gene was not expressed in the uninduced or induced JCS cells. Our results suggest that IL-1alpha, IL-1beta and IL-4 may act on the later stage of biochanin A-mediated differentiation of JCS cells.