Triptolide induces apoptotic cell death of multiple myeloma cells via transcriptional repression of Mcl-1.

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Quinone methide tripterine, celastrol, induces apoptosis in human myeloma cells via NF-κB pathway.

Multiple myeloma is still an incurable hematological malignancy despite the development of high-dose chemotherapy with stem cell transplantation. However, the therapeutic approach for multiple myeloma has progressed significantly in the last decade. Novel agents such as bortezomib, thalidomide and lenalidomide have been introduced in clinics as expanded treatment options and have improved the outcomes of patients with multiple myeloma. More recently, the development of novel agents with better effects and lower side-effects for the treatment of multiple myeloma has became necessary in the clinical setting. Celastrol is a quinone methide triterpene derived from the medicinal plant Tripterygium wilfordii, which has been used to treat chronic inflammatory and autoimmune diseases. It also has been reported that celastrol has potential as an anticancer agent; however, the effects of celastrol against myeloma have never been reported. It has been reported that the mechanisms of action occur via the NF-κB pathway. However, the effects of celastrol against multiple myeloma have never been reported. The recent clinical success of proteasome inhibitor bortezomib, which acts by inhibiting the NF-κB activity in patients with multiple myeloma led us to investigate the effects of celastrol on myeloma cells. Here we found for the first time that celastrol induces cell cycle arrest at the G1 phase followed by apoptosis in human myeloma cell line U266 cells. In addition, we showed that celastrol induces apoptosis of myeloma cells via activation of the caspase-3 and NF-κB pathways. These results suggest that celastrol would be an effective therapeutic agent in signal transduction therapy for the treatment of patients with multiple myeloma.

Cantharidin induces apoptosis of human multiple myeloma cells via inhibition of the JAK/STAT pathway.

Multiple myeloma is an incurable B-cell malignancy requiring new therapeutic strategies in clinical settings. Interleukin (IL)-6 signaling pathways play a critical role in the pathogenesis of multiple myeloma. The traditional Chinese medicine cantharidin (CTD) has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of CTD as a novel therapeutic agent for the patients with multiple myeloma. We investigated the in vitro effects of CTD for its antimyeloma activity, and further examined the molecular mechanisms of CTD-induced apoptosis. CTD inhibited the cellular growth of human myeloma cell lines as well as freshly isolated myeloma cells in patients. Cultivation with CTD induced apoptosis of myeloma cells in a cell-cycle-independent manner. Treatment with CTD induced caspase-3, -8, and -9 activities, and it was completely blocked by each caspase inhibitor. We further examined the effect of CTD on the IL-6 signaling pathway in myeloma cells, and found that CTD inhibited phosphorylation of STAT3 at tyrosine 705 residue as early as 1 h after treatment and down-regulated the expression of the antiapoptotic bcl-xL protein. STAT3 directly bound and activated the transcription of bcl-xL gene promoter, resulting in the induction of the expression of bcl-xL in myeloma cells. The essential role of STAT3 in CTD effects was confirmed by transfection with the constitutively active and dominant negative form of STAT3 in U266 cells. In conclusion, we have demonstrated that CTD is a promising candidate to be a new therapeutic agent in signal transduction therapy.

Compound Danshen injection improves endotoxin-induced microcirculatory disturbance in rat mesentery.

AIM: To investigate the effect of compound Danshen injection on lipopolysaccharide (LPS)-induced rat mesenteric microcirculatory dysfunctions and the underlying possible mechanism by an inverted intravital microscope and high-speed video camera system. METHODS: LPS was continuously infused through the jugular artery of male Wistar rats at the dose of 2 mg/kg per hour. Changes in mesenteric microcirculation, such as diameters of arterioles and venules, velocity of RBCs in venules, leukocyte rolling, adhesion and emigration, free radicals released from post-capillary venules, FITC-albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope assisted with CCD camera and SIT camera. Meanwhile, the expression of adhesion molecules CD11b/CD18 and the production of free radical in neutrophils, and the expression of intercellular adhesion molecule 1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) were quantified by flow cytometry (FACS) in vitro. RESULTS: The continuous infusion with LPS resulted in a number of responses in microcirculation, including a significant increase in the positive region of venule stained with Monastral blue B, rolling and adhesion of leukocytes, production of oxygen radical in venular wall, albumin efflux and enhanced mast cell degranulation in vivo, all of which, except for the leukocyte rolling, were attenuated by the treatment with compound Danshen injection. Experiments performed in vitro further revealed that the expression of CD11b/CD18 and the production of oxygen free radical in neutrophils, and the expression of ICAM-1 in HUVECs were increased by exposure to LPS, and they were attenuated by compound Danshen injection. CONCLUSION: These results suggest that compound Danshen injection is an efficient drug with multi-targeting potential for improving the microcirculatory disturbance.

Emodin has a cytotoxic activity against human multiple myeloma as a Janus-activated kinase 2 inhibitor.

Emodin is an active component of a traditional Chinese and Japanese medicine isolated from the root and rhizomes of Rheum palmatum L. Here, we show that emodin significantly induces cytotoxicity in the human myeloma cells through the elimination of myeloid cell leukemia 1 (Mcl-1). Emodin inhibited interleukin-6-induced activation of Janus-activated kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3), followed by the decreased expression of Mcl-1. Activation of caspase-3 and caspase-9 was triggered by emodin, but the expression of other antiapoptotic Bcl-2 family members, except Mcl-1, did not change in the presence of emodin. To clarify the importance of Mcl-1 in emodin-induced apoptosis, the Mcl-1 expression vector was introduced into the human myeloma cells by electroporation. Induction of apoptosis by emodin was almost abrogated in Mcl-1-overexpressing myeloma cells as the same level as in parental cells, which were not treated with emodin. In conclusion, emodin inhibits interleukin-6-induced JAK2/STAT3 pathway selectively and induces apoptosis in myeloma cells via down-regulation of Mcl-1, which is a good target for treating myeloma. Taken together, our results show emodin as a new potent anticancer agent for the treatment of multiple myeloma patients.

1'-Acetoxychavicol acetate induces apoptosis of myeloma cells via induction of TRAIL.

A component of a traditional Thai condiment, 1'-acetoxychavicol acetate (ACA), is a natural compound, and it is obtained from rhizomes of the ethno-medicinal plant Languas galanga (Zingiberaceae). Our previous studies showed that ACA dramatically inhibited cellular growth of multiple myeloma cells in vivo and in vitro through the induction of apoptosis in association with the activation of caspase-8, inactivation of NF-kappaB, and down-regulation of anti-apoptotic proteins. Subsequently, we investigated the detailed apoptotic pathway of ACA and further demonstrated that ACA up-regulates the expression of both TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) and TRAIL receptor death receptor 5 (DR5). In addition, TRAIL/R-Fc chimera neutralizes the ACA-induced apoptosis. These results suggest that the death signaling of TRAIL is involved in the ACA-induced apoptosis of myeloma cells, and provide a rationale for the induction of TRAIL/Apo2L by ACA, which could potentially be used as a novel therapeutic agent in patients with multiple myeloma.

Green tea component, catechin, induces apoptosis of human malignant B cells via production of reactive oxygen species.

PURPOSE: Green tea polyphenol, (-)-epigallocatechin-3-gallate, has been shown to inhibit cellular proliferation and induce apoptosis of various cancer cells. The aim of this study was to investigate the possibility of (-)-epigallocatechin-3-gallate as a novel therapeutic agent for the patients with B-cell malignancies including multiple myeloma. EXPERIMENTAL DESIGN: We investigated the effects of (-)-epigallocatechin-3-gallate on the induction of apoptosis in HS-sultan as well as myeloma cells in vitro and further examined the molecular mechanisms of (-)-epigallocatechin-3-gallate-induced apoptosis. RESULTS: (-)-Epigallocatechin-3-gallate rapidly induced apoptotic cell death in various malignant B-cell lines in a dose- and time-dependent manner. (-)-Epigallocatechin-3-gallate-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials (Deltapsim); the release of cytochrome c, Smac/DIABLO, and AIF from mitochondria into the cytosol; and the activation of caspase-3 and caspase-9. Elevation of intracellular reactive oxygen species (ROS) production was also shown during (-)-epigallocatechin-3-gallate-induced apoptosis of HS-sultan and RPMI8226 cells as well as fresh myeloma cells. Antioxidant, catalase, and Mn superoxide dismutase significantly reduced ROS production and (-)-epigallocatechin-3-gallate-induced apoptosis, suggesting that ROS plays a key role in (-)-epigallocatechin-3-gallate-induced apoptosis in B cells. Furthermore, a combination with arsenic trioxide (As2O3) and (-)-epigallocatechin-3-gallate significantly enhanced induction of apoptosis compared with As2O3 alone via decreased intracellular reduced glutathione levels and increased production of ROS. CONCLUSIONS: (-)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. In addition, (-)-epigallocatechin-3-gallate enhanced As2O3-induced apoptosis in human multiple myeloma cells.

1'-acetoxychavicol acetate is a novel nuclear factor kappaB inhibitor with significant activity against multiple myeloma in vitro and in vivo.

1'-Acetoxychavicol acetate (ACA) is a component of a traditional Asian condiment obtained from the rhizomes of the commonly used ethno-medicinal plant Languas galanga. Here, we show for the first time that ACA dramatically inhibits the cellular growth of human myeloma cells via the inhibition of nuclear factor kappaB (NF-kappaB) activity. In myeloma cells, cultivation with ACA induced G0-G1 phase cell cycle arrest, followed by apoptosis. Treatment with ACA induced caspase 3, 9, and 8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we showed that ACA significantly inhibits the serine phosphorylation and degradation of IkappaBalpha. ACA rapidly decreased the nuclear expression of NF-kappaB, but increased the accumulation of cytosol NF-kappaB in RPMI8226 cells, indicating that ACA inhibits the translocation of NF-kappaB from the cytosol to the nucleus. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight significantly decreased in the ACA-treated mice compared with the control mice. In conclusion, ACA has an inhibitory effect on NF-kappaB, and induces the apoptosis of myeloma cells in vitro and in vivo. ACA, therefore, provides a new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-kappaB inhibitor.

Induction of apoptosis in human myeloid leukemic cells by 1'-acetoxychavicol acetate through a mitochondrial- and Fas-mediated dual mechanism.

PURPOSE: The purpose of this investigation was to determine the antileukemic effects of 1'-acetoxychavicol acetate (ACA) obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberaceae). EXPERIMENTAL DESIGN: We evaluated the effects of ACA on various myeloid leukemic cells in vitro and in vivo. We further examined the molecular mechanisms of ACA-induced apoptosis in myeloid leukemic cells. RESULTS: Low-dose ACA dramatically inhibited cellular growth of leukemic cells by inducing apoptosis. Because NB4 promyelocytic leukemic cells were most sensitive to ACA, we used NB4 cells for further analyses. Production of reactive oxygen species triggered ACA-induced apoptosis. ACA-induced apoptosis in NB4 cells was in association with the loss of mitochondrial transmembrane potential (DeltaPsim) and activation of caspase-9, suggesting that ACA-induced death signaling is mediated through a mitochondrial oxygen stress pathway. In addition, ACA activated Fas-mediated apoptosis by inducing of casapse-8 activity. Pretreatment with the thiol antioxidant N-acetyl-L-cysteine (NAC) did not inhibit caspase-8 activation, and the antagonistic anti-Fas antibody ZB4 did not block generation of reactive oxygen species, indicating that both pathways were involved independently in ACA-induced apoptosis. Furthermore, ACA had a survival advantage in vivo in a nonobese diabetic/severe combined immunodeficient mice leukemia model without any toxic effects. CONCLUSIONS: We conclude that ACA induces apoptosis in myeloid leukemic cells via independent dual pathways. In addition, ACA has potential as a novel therapeutic agent for the treatment of myeloid leukemia.

A novel therapeutic approach for hematological malignancies based on cellular differentiation and apoptosis.

Hematological malignancies including acute leukemia, and multiple myeloma are disorders characterized by the accumulation of neoplastic hematopoietic cells, resulting in aggressive clinical manifestations with poor prognosis. The therapeutic approach to these disorders is basically chemotherapy for achieving complete remission based on the concept of total cell kill. However, severe side effects and complications such as serious infection and bleeding due to anti-cancer drugs are major problems in the clinical setting. In addition, repeated episodes of relapse of the disease may lead to refractory or chemotherapy-resistant disorders. These problems are occurred because anti-cancer agents have effects on both cancer cells and normal hematopoietic cells. The clinical evidences thus suggest the limitations of the chemotherapy for hematological malignancies: novel effective therapeutic approaches with less toxicity are therefore actively being sought. Differentiation-inducing therapy employing a physiologically active derivative of vitamin A, all-trans retinoic acid (ATRA), brought remarkably advances in the therapeutic outcome of APL at the end of last century. More recently, the clinical success of imatinib mesylate (STI571), potent competitive inhibitor of the Bcr/Abl protein tyrosine kinase, in the treatment of CML has focused enthusiasm toward molecular targeted therapy for the hematological malignancies. The therapeutic activity of these agents can be explained by their abilities to modify cellular growth, differentiation, and apoptosis in cells by activating unknown gene programs that molecular cellular proliferation. We have actively sought out new agents among natural products and cytokines with the ability to induce cellular differentiation and apoptosis. In this symposium, I will present our recent data of these novel compounds and their molecular mechanisms for inducing differentiation and apoptosis of hematological malignant cells.

A novel therapeutic technology of specific RNA inhibition for acute promyelocytic leukemia: improved design of maxizymes against PML/RARalpha mRNA.

Targeting of PML/RARalpha using a loss of function strategy in acute promyelocytic leukemia (APL) is a direct therapeutic approach for patients and may be the basis of future gene therapy for this leukemia. To achieve this, we designed specific maxizymes, novel allosterically controllable ribozymes, against both short and long PML/RARalpha isoforms. The maxizyme has sensor arms that can only recognize target sequences, and it can form a cavity that captures catalytically indispensable Mg2+. We deleted 1 base nucleotide in the Mg2+-binding pocket designed MzPRT50 and MzPRK55. The distance from the PML/RARalpha junction site to the center of effectors is only 2 bases, and there are 8 and 9 complementary bases in their inactive forms, respectively. Both maxizymes specifically cleaved PML/RARalpha mRNA but not wild-type RARalpha mRNA in a cell-free system. Modification of the sequence of the Mg2+-binding pocket will be important in designing the sequence-specific maxizymes against oncogenic genes.