Yongkui Jing

Boswellic acid acetate induces differentiation and apoptosis in leukemia cell lines

Boswellic acid acetate (BC-4), a compound isolated from the herb Boswellia carterii Birdw., can induce differentiation and apoptosis of leukemia cells. Based on cell morphology and NBT reduction, BC-4 induced monocytic differentiation of myeloid leukemia HL-60, U937 and ML-1 cells at a dose under 12.5 microg/ml (24.2 microM). BC-4 was a potent inducer, with 90% of the cells showing morphologic changes and 80-90% of the cells showing NBT reduction. Specific and non-specific esterase were also increased by BC-4. Based on benzidine staining assay, BC-4 failed to induce erythroid leukemia DS-19 and K562 cells differentiation. In contrast to its selective differentiation effect, BC-4 strongly inhibited growth of all cell lines tested. The growth inhibition effect was dose- and time-dependent. In HL-60 cells, 20 microg/ml (38.8 microM) of BC-4 decreased viable cell number by 60% at 24 h, whereas at 3 days there was virtually no viable cells. Morphologic and DNA fragmentation analysis proved that BC-4 induced cell apoptosis. The dual apoptotic and differentiation effects of BC-4 suggest that it may be a powerful agent in the treatment of leukemia.

Acetyl-Keto-β-Boswellic Acid Induces Apoptosis through a Death Receptor 5–Mediated Pathway in Prostate Cancer Cells

Acetyl-keto-beta-boswellic acid (AKBA), a triterpenoid isolated from Boswellia carterri Birdw and Boswellia serrata, has been found to inhibit tumor cell growth and to induce apoptosis. The apoptotic effects and the mechanisms of action of AKBA were studied in LNCaP and PC-3 human prostate cancer cells. AKBA induced apoptosis in both cell lines at concentrations above 10 microg/mL. AKBA-induced apoptosis was correlated with the activation of caspase-3 and caspase-8 as well as with poly(ADP)ribose polymerase (PARP) cleavage. The activation of caspase-8 was correlated with increased levels of death receptor (DR) 5 but not of Fas or DR4. AKBA-induced apoptosis, caspase-8 activation, and PARP cleavage were inhibited by knocking down DR5 using a small hairpin RNA. AKBA treatment increased the levels of CAAT/enhancer binding protein homologous protein (CHOP) and activated a DR5 promoter reporter but did not activate a DR5 promoter reporter with the mutant CHOP binding site. These results suggest that AKBA induces apoptosis in prostate cancer cells through a DR5-mediated pathway, which probably involves the induced expression of CHOP.

Buthionine Sulfoximine Enhancement of Arsenic Trioxide-Induced Apoptosis in Leukemia and Lymphoma Cells Is Mediated via Activation of c-Jun NH2-Terminal Kinase and Up-regulation of Death Receptors

The mechanism of apoptosis induced by treatment with As(2)O(3) alone or in combination with buthionine sulfoximine (BSO) was studied in NB4, U937, Namalwa, and Jurkat cells. As(2)O(3) at concentrations <2 micromol/L induced apoptosis in NB4 cells and Namalwa cells but not in U937 and Jurkat cells. As(2)O(3)-induced apoptosis in NB4 cells and Namalwa cells correlated with increase of H(2)O(2) and caspase activation without activation of c-Jun NH(2)-terminal kinase (JNK). BSO (10 micromol/L) depleted the reduced form of intracellular glutathione without inducing apoptosis but synergized with 1 micromol/L As(2)O(3) to induce apoptosis in all four cell lines. This synergy correlated with JNK activation. Treatment with As(2)O(3) plus BSO, but not with As(2)O(3) alone, increased the levels of death receptor (DR) 5 protein and caspase-8 cleavage. The JNK inhibitor SP600125 inhibited the increase in DR5 protein and attenuated apoptosis induced by treatment with As(2)O(3) plus BSO. These observations suggest that a DR-mediated pathway activated by JNK is involved in apoptosis induced by treatment with As(2)O(3) plus BSO.

Boswellic acid acetate induces apoptosis through caspase-mediated pathways in myeloid leukemia cells

The mechanism of the cytotoxic effect of boswellic acid acetate, a 1:1 mixture of α-boswellic acid acetate and β-boswellic acid acetate, isolated from Boswellia carterri Birdw on myeloid leukemia cells was investigated in six human myeloid leukemia cell lines (NB4, SKNO-1, K562, U937, ML-1, and HL-60 cells). Morphologic and DNA fragmentation assays indicated that the cytotoxic effect of boswellic acid acetate was mediated by induction of apoptosis. More than 50% of the cells underwent apoptosis after treatment with 20 μg/mL boswellic acid for 24 hours. This apoptotic process was p53 independent. The levels of apoptosis-related proteins Bcl-2, Bax, and Bcl-XL were not modulated by boswellic acid acetate. Boswellic acid acetate induced Bid cleavage and decreased mitochondrial membrane potential without production of hydrogen peroxide. A general caspase inhibitor (Z-VAD-FMK) and a specific caspase-8 inhibitor II (Z-IETD-FMK) blocked boswellic acid acetate–induced apoptosis. The mRNAs of death receptors 4 and 5 (DR4 and DR5) were induced in leukemia cells undergoing apoptosis after boswellic acid acetate treatment. These data taken together suggest that boswellic acid acetate induces myeloid leukemia cell apoptosis through activation of caspase-8 by induced expression of DR4 and DR5, and that the activated caspase-8 either directly activates caspase-3 by cleavage or indirectly by cleaving Bid, which in turn decreases mitochondria membrane potential.

Downregulation of Mcl-1 through GSK-3β activation contributes to arsenic trioxide-induced apoptosis in acute myeloid leukemia cells

Arsenic trioxide (ATO) induces disease remission in acute promyelocytic leukemia (APL) patients, but not in non-APL acute myeloid leukemia (AML) patients. ATO at therapeutic concentrations (1–2 μM) induces APL NB4, but not non-APL HL-60, cells to undergo apoptosis through the mitochondrial pathway. The role of antiapoptotic protein Mcl-1 in ATO-induced apoptosis was determined. The levels of Mcl-1 were decreased in NB4, but not in HL-60, cells after ATO treatment through proteasomal degradation. Both glycogen synthase kinase-3β (GSK-3β) inhibitor SB216763 and siRNA blocked ATO-induced Mcl-1 reduction as well as attenuated ATO-induced apoptosis in NB4 cells. Silencing Mcl-1 sensitized HL-60 cells to ATO-induced apoptosis. Both ERK and AKT inhibitors decreased Mcl-1 levels and enhanced ATO-induced apoptosis in HL-60 cells. Sorafenib, an Raf inhibitor, activated GSK-3β by inhibiting its phosphorylation, decreased Mcl-1 levels and decreased intracellular glutathione levels in HL-60 cells. Sorafenib plus ATO augmented reactive oxygen species production and apoptosis induction in HL-60 cells and in primary AML cells. These results indicate that ATO induces Mcl-1 degradation through activation of GSK-3β in APL cells and provide a rationale for utilizing ATO in combination with sorafenib for the treatment of non-APL AML patients.

Upregulation of Bfl-1/A1 in leukemia cells undergoing differentiation by all-trans retinoic acid treatment attenuates chemotherapeutic agent-induced apoptosis.

All-trans retinoic acid (ATRA) induces differentiation of NB4 and HL-60 leukemia cells, but not R4 and HL-60/Res cells. Three agents used in cancer therapy, doxorubicin (Dox), arsenic trioxide (As2O3) and paclitaxel, induce apoptosis, but not differentiation, in all of these cell lines. The induction of apoptosis by these agents is decreased in ATRA-pretreated NB4 and HL-60 cells, but not in ATRA-pretreated R4 and HL-60/Res cells. The level of Bcl-2 protein is decreased by ATRA treatment in NB4, HL-60 and HL-60/Res cells. The level of Mcl-1 protein is increased by ATRA treatment in NB4 and R4 cells, but not in HL-60 and HL-60/Res cells. Bfl-1/A1 mRNA is not expressed in these cell lines, however, its expression is markedly induced by ATRA treatment in NB4 and HL-60 cells, but not in R4 or HL-60/Res cells, which correlates with inhibition of apoptosis. Inhibiting Bfl-1/A1 mRNA upregulation in ATRA-pretreated NB4 cells using small interfering RNA (siRNA) partly recovers cell sensitivity to Dox-induced apoptosis. These data demonstrate that ATRA induction of Bfl-1/A1 in differentiated NB4 and HL-60 cells contributes to a loss of sensitivity to chemotherapy-induced apoptosis.

Defective expression of cellular retinol binding protein type I and retinoic acid receptors alpha2, beta2, and gamma2 in human breast cancer cells.

Because the retinoic acid (RA) signaling pathway regulates cell proliferation and differentiation, inactivation of genes integral to the pathway represents a potential mechanism of carcinogenesis. We have studied in human breast cancer cells (T47D, MCF‐7, ZR75‐1, MDA‐MB‐231, and BT20) the expression of a subset of retinoid signaling genes that are themselves transcriptionally up‐regulated by RA, the cellular retinol binding protein type I (CRBPI) and the RA receptors (RARs) (α2, β2, and ǵ2. We find that constitutive expression of these genes is low or undetectable, and that expression levels are seldom responsive to 24 h treatment with 1 μM all‐trans or 9‐cis RA (Northern blot analysis). This is in contrast to breast fibroblasts, which show RA‐de‐ pendent expression of all four genes under the same conditions. Moreover, normal human breast epi‐thelial cells express CRBPI and RARβ2 at the mRNA level, suggesting that loss of expression of these genes is tied to malignant transformation. RARβ2, but not CRBPI, was also expressed in RA‐treated MTSV1‐7 cells, an immortalized but nontumorigenic luminal epithelial cell line. Lack of CRBPI and RARβ2 expression in cancer cells was not due to general impairment of RA signaling, as shown by RA activation of a RARE3‐tk‐CAT reporter in a subclone of MDA‐MB‐231 cells that did not express either CRBPI or RARβ2. These results suggest that at least two independent defects in the expression of proteins that function in retinoid signaling may be involved in breast carcinogenesis.—Jing, Y., Zhang, J., Blei‐ weiss, I. J., Waxman, S., Zelent, A., and Mira‐y‐ Lopez, R. Defective expression of cellular retinol binding protein type I and retinoic acid receptors a2, β2, and ǵ2 in human breast cancer cells. FASEB J. 10,1064‐1070 (1996)

Dual effects of glutathione-S-transferase π on As2O3 action in prostate cancer cells: enhancement of growth inhibition and inhibition of apoptosis

To determine the effects of glutathione-S-transferase π (GSTπ) on the actions of As2O3, As2O3-induced growth inhibition and apoptosis was studied in three prostate cancer cell lines: DU-145, PC-3 and LNCaP cells. As2O3 inhibited cell proliferation of DU-145 and PC-3 cells (both cells express GSTπ), but not of LNCaP cells (which lack GSTπ expression) at concentrations below 1 μM. LNCaP cells stably transfected and expressed GSTπ (LNCaP/GSTπ) became sensitive to As2O3 growth inhibition. As2O3 arrested cell growth of DU-145, PC-3 and LNCaP/GSTπ cells in the G2/M phase of the cell cycle at low concentrations (<2 μM), but did not induce apoptosis. At higher concentrations (10–20 μM), As2O3 induced apoptosis in LNCaP cells, but not in DU-145 or PC-3 cells. The apoptosis induction due to As2O3 treatment of LNCaP cell correlated with the activation of JNK and p38 and induction of p53 protein. LNCaP/GSTπ cells became insensitive to As2O3-induced apoptosis with reduced JNK activition. These data indicate that GSTπ increases growth inhibition due to As2O3 treatment and prevents As2O3-induced apoptosis in prostate cancer cells. Therefore, it appears that As2O3 inhibits cell growth and induces apoptosis through different mechanisms.

The cleavage product ΔPML-RARα contributes to all-trans retinoic acid-mediated differentiation in acute promyelocytic leukemia cells

PML–RARα protein, the leukemogenic product of t(15,17) in acute promyelocytic leukemia, is cleaved into a truncated form termed ΔPML–RARα during all-trans retinoic acid (ATRA)-induced differentiation of NB4 cells. ΔPML–RARα is not formed in ATRA differentiation resistant NB4 subclones. As2O3 inhibits ΔPML–RARα formation and differentiation-induction when given in combination with ATRA. Treatment with hexamethylene bisacetamide (HMBA) combined with ATRA enhances ATRA-induced differentiation in ATRA-insensitive NB4-CI and arsenic-resistant NB4/As cells, and is associated with stabilization of PML–RARα protein and increased ΔPML–RARα formation. Unlike forced expression of PML–RARα, forced ΔPML–RARα expression based on an estimated deletion of the N-terminal PML portion does not repress RARE-tk-luc reporter activity mediated by endogenous retinoic acid receptors. The cleavage of PML–RARα is blocked by RARα antagonist Ro-41-5253 and cycloheximide and therefore requires a RARα transactivation-dependent pathway. Proteasome inhibitor MG-132 and caspase inhibitor Z-VAD-FMK do not block ATRA-induced PML–RARα cleavage and differentiation. These data suggest that (a) ATRA treatment induces PML–RARα cleavage by induction of unknown enzymes independent of proteasome- and caspase-mediated pathways; (b) ΔPML–RARα might function differently from both PML–RARα and RARα; (c) failure to cleave PML–RARα and form ΔPML–RARα after ATRA treatment may contribute to ATRA resistance in APL cells.