Amooranin (AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A. rohituka stem bark is one of the components of a medicinal preparation used in the Indian Ayurvedic system of medicine for the treatment of human malignancies. We investigated the mechanism of cell death associated with AMR cytotoxicity in human mammary carcinoma MCF-7, multidrug resistant breast carcinoma MCF-7/TH and breast epithelial MCF-10A cell lines. AMR IC50 values ranged between 3.8-6.9 microg/ml among MCF-7, MCF-7/TH and MCF-10A cells. AMR induced oligonucleosome-sized DNA ladder formation characteristic of apoptosis when tumor cells were treated with 1-8 microg/ml AMR for 48 h. In situ cell death detection assay indicated that AMR caused 37.3-72.1% apoptotic cells in MCF-7, 32-48.7% in MCF-7/TH and 0-37.1% in MCF- 10A cells at 1-8 microg/ml concentrations. The induction of apoptosis in AMR treated cells was accompanied by the elevation of total caspase and caspase-8 activities. Flow cytometric analysis showed that AMR induced caspase-8 activation in 40.8-71% MCF-7, 28.5-43.2% MCF-7/TH and 4-32.8% MCF-10A cells at 1-8 microg/ml concentrations. Our results suggest that AMR is a novel drug having potential for clinical development against human malignancies.
A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.
Amooranin (AMR), a natural triterpenoid drug isolated and characterized from Amoora rohituka stem bark, is cytotoxic to SW620 human colon carcinoma cell line with an IC 50 value of 2.9 lg/ml. This novel compound caused depolarization of mitochondrial membrane and decrease of membrane potential, indicating initial signal of apoptosis induction. The percentage of cells with decreased mitochondrial potential ranged from 7.4% at 1 lg/ml to 60.5% at 100 lg/ml AMR. Flow cytometric analysis of apoptosis using Annexin-V-FITC staining showed that the percentage of apoptotic cells ranged from 7.5% at 1 lg/ml to 59.2% at 100 lg/ml AMR. AMR-induced apoptosis was accompanied by redistribution of cytochrome c from mitochondria to cytosol as well as down regulation of Bcl-2 and Bcl-X L proteins in a dose-dependent manner. SW620 human colon carcinoma xenograft mice treated with AMR showed significant reduction in tumor growth rates compared to saline-and doxorubicin-treated groups. The reduction in tumor growth rate was better in xenografts treated with 2 mg/kg AMR than 5 and 10 mg/kg treated mice. The analysis of global gene expression changes induced by AMR in xenograft tumors by microarray hybridization revealed that several genes involved in energy pathways, transport, apoptosis, immune response, nucleic acid metabolism, protein metabolism, cell growth and/or maintenance, signal transduction and cell communication, were affected by this natural cancer drug. These results suggest that the anticancer properties of AMR in SW620 human colon carcinoma cell line are mediated through its effects on functional genomics, targeting the apoptotic process. ' 2006 Wiley-Liss, Inc.
An alpha-D-glucan (RR1) composed of (1-->4) linked back bone and (1-->6) linked branches with a molecular mass of >550 kDa and exhibiting unique immune stimulating properties is isolated and characterized from the medicinal plant Tinospora cordifolia. This novel polysaccharide is noncytotoxic and nonproliferating to normal lymphocytes as well as tumor cell lines at 0-1000 microg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 microg/ml concentration. The significant activation of NK cells is associated with the dose-dependent killing of tumor cells by activated normal lymphocytes in a functional assay. Immune activation by RR1 in normal lymphocytes elicited the synthesis of interleukin (IL)-1beta (1080 pg/ml), IL-6 (21,833 pg/ml), IL-12 p70 (50.19 pg/ml), IL-12 p40 (918.23 pg/ml), IL-18 (27.47 pg/ml), IFN- gamma (90.16 pg/ml), tumor necrosis factor (TNF)-alpha (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 microg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-alpha and TNF-beta. The cytokine profile clearly demonstrates the Th1 pathway of T helper cell differentiation essential for cell mediated immunity, with a self-regulatory mechanism for the control of its overproduction. RR1 also activated the complements in the alternate pathway, demonstrated by a stepwise increase in C3a des Arg components. Incidentally, RR1 stimulation did not produce any oxidative stress or inducible nitric oxide synthase (iNOS) in the lymphocytes or any significant increase in nitric oxide production. The water solubility, high molecular mass, activation of lymphocytes especially NK cells, complement activation, Th1 pathway-associated cytokine profile, together with a low level of nitric oxide synthesis and absence of oxidative stress confer important immunoprotective potential to this novel alpha-D-glucan.
A newborn baby boy was diagnosed with the mixed form of congenital mesoblastic nephroma (CMN) representing both classic and cellular histology features in the renal tumor. Additionally, the patient had skin and bone lesions consistent with multifocal involvement of a generalized infantile fibromatosis (IFS). Both skin and bone lesions were distinctly different from CMN and did not represent metastasis. The primary tumor cell line (MCH-MN-1), established from the resected right kidney tumor, had a diploid DNA content. Cytogenetic studies revealed deletion on the long arm of chromosome 3 (q21q24) and duplication on the short arm of chromosome 11 (p15). MCH-MN-1 cells expressed ETV6-NTRK3 gene fusion transcripts, characteristic of cellular and mixed forms of CMNs. The cells had high p21 and low Bax mRNA expression in the reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The high level of proliferative marker (Ki67) mRNA expression correlated well with the pluripotent nature of MCH-MN-1 in tissue culture (cell doubling time = 12.4 h). Our results showed that MCH-MN-1 might be a good model cell line for investigations on mesoblastic nephroma.
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