phenanthrenes and Multiple-Myeloma

To explore the effects of micro ribonucleic acid (miR)-32 on the proliferation and apoptosis of myeloma cells, and to verify whether it exerts its function by targeting phosphatase and tensin homolog deleted on chromosome ten (PTEN).. The differentially expressed miRNAs were screened in healthy people and myeloma patients. The myeloma U266 cells transfected with negative control (NC) were used as control group, those transfected with miR-32 inhibitor as transfection group, and those transfected with miR-32 inhibitor and treated with PTEN inhibitor SF1670 as the transfection + inhibitor group. Then, the cell proliferation and apoptosis in each group were detected using the 5-Ethynyl-2'-deoxyuridine (EdU) kit and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Finally, the expressions of apoptosis-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2 homologous antagonist/killer (Bak), caspase-9, and survivin were detected.. The expressions of some miRNAs and genes in myeloma patients were significantly different from those in healthy people. In myeloma patients, miR-32, miR-126, miR-123, and miR-183 were significantly highly expressed, while miR-5, miR-76, and miR-50 were remarkably lowly expressed. After myeloma U266 cells were transfected with the miR-32 inhibitor, the expression of miR-32 markedly declined. In addition, the mRNA expression of PTEN in myeloma cells rose after transfection with the miR-32 inhibitor, and declined after addition of the PTEN inhibitor SF1670, which were consistent with the results of Western blotting. Besides, the proliferation ability of myeloma cells was evidently weakened after transfection with the miR-32 inhibitor, while it was restored to a certain extent after addition of the PTEN inhibitor SF1670. Moreover, the number of apoptotic myeloma cells was remarkably larger after transfection with the miR-32 inhibitor, while it was remarkably smaller after addition of the PTEN inhibitor SF1670. The expressions of pro-apoptotic proteins Bak and caspase-9 in myeloma cells were significantly increased after transfection with the miR-32 inhibitor (p<0.05), and significantly decreased after addition of the PTEN inhibitor SF1670, while the expressions of anti-apoptotic proteins Bcl-2 and survivin were opposite to those of Bak and caspase-9.. MiR-32 targeting PTEN will have certain effects on the proliferation and apoptosis of myeloma cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; MicroRNAs; Multiple Myeloma; Phenanthrenes; PTEN Phosphohydrolase

To investigate the effect of triptolide(TPL) on proliferation and apoptosis of RPMI8226 cells and its mechanism.. MTT assay was used to measure the proliferation of RPMI8226 cells after treatment with different concentration (10, 20, 40, 80 and 160 nmol/L) of TPL for different incubation time (24 h, 48 h and 72 h). The cell apoptosis was detected by flow cytometry, the mRNA expressions of SMYD3 and MMP-9 were measured by quantitative real-time PCR, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells was assayed by Western blot.. TPL inhibited RPMI8226 cell proliferation, and the inhibitory rate of cell proliferation increased significantly in a dose- and time-dependent manner(P<0.05), the RPMI8226 cell apoptosis was induced by treatment with 40, 80 and 160 nmol/L TPL (P<0.05), the qRT-PCR showed that treatment of RPMI8226 cells with TPL down-regulated the mRNA expression of SMYD3 in a dose-dependent manner(P<0.05). Compared with the blank group, the mRNA expression level of MMP-9 in RPMI8226 cells transfected by siRNA-SMYD3 was significantly depressed. Western blot showed that the protein levels of H3K4me2 and H3K4me3 were decreased in a dose-dependent manner after TPL treatment(P<0.05). Compared with the blank group and siRNA negative group, the protein level of H3K4me2 and H3K4me3 in RPMI8226 cells transfected by siRNA-SMYD3 also were significantly depressed(P<0.05).. TPL can significantly inhibit the proliferation of RPMI8226 cells and induce their apoptosis, which may be related to the inhibition of SMYD3 expression by TPL- down-regulating the H3K4 methylation and the activating the MMP-9 transcription.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Diterpenes; Epoxy Compounds; Histone-Lysine N-Methyltransferase; Humans; Matrix Metalloproteinase 9; Multiple Myeloma; Phenanthrenes

Triptolide, an active component extracted from the medicinal plant Tripterygium wilfordii Hook F., has been used to treat various diseases, including lupus, cancer, rheumatoid arthritis and nephritic syndrome. The present study investigated the effects of triptolide on multiple myeloma using western blotting and an electrophoretic mobility shift assay. Triptolide was found to suppress the inducible and constitutive activation of signal transducer and activator of transcription 3 (STAT3), which is closely associated with inflammation and tumorigenesis. Triptolide also inhibited the DNA binding of STAT3. This correlated with the downregulation of Src kinase and Janus kinase 1 and 2, and with the upregulation of protein tyrosine phosphatase non‑receptor type 6 (also known as SHP‑1). In addition, triptolide downregulated the expression of the STAT3‑regulated antiapoptotic (Bcl‑xL and myeloid cell leukemia‑1), proliferative (cyclin D1), and angiogenic (vascular endothelial growth factor) genes, suggesting that triptolide can induce apoptosis of tumor cells. These results suggest that triptolide may be a potential therapeutic anticancer agent for the prevention and treatment of multiple myeloma; thus further in‑depth investigations into its efficacy and toxicity are warranted.

Topics: Antineoplastic Agents, Alkylating; Cell Line, Tumor; Diterpenes; Dose-Response Relationship, Drug; Enzyme Activation; Epoxy Compounds; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 1; Janus Kinase 2; Multiple Myeloma; Phenanthrenes; Phosphorylation; Protein Binding; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor

There is still no ideal treatment for multidrug resistant multiple myeloma, looking for drugs which can reverse chemotherapy resistance and enhance curative effects of chemotherapy drugs becomes a problem that needs to be solved urgently. Poly(ADP-ribose) polymerase inhibitors appear to be an important tool for medical therapy of several malignancies. In the present study, we investigated the potential of the PARP-1 inhibitor PJ34, in vitro, to further enhance the efficacy of the traditional chemotherapy drug melphalan in the multidrug-resistant multiple myeloma cell line RPMI8226/R. The effects of different concentrations of PJ34 and melphalan on cell proliferation were determined by the CCK-8 assay. The expressions of FA/BRCA pathway-related factors were detected by western blotting and RT-PCR. The percentage of cell apoptosis was measured with flow cytometry. DNA double-strand break (DSB) repair was quantified by γH2AX immunofluorescence. In addition, DNA damage repair at the level of the individual cell was determined by comet assay. Co-administration of PJ34 and melphalan had synergistic inhibitory effects on the proliferation of RPMI8226/R cells, suggesting more powerful antitumor activities. The apoptosis percentage also was increased more obviously by the treatment of melphalan plus PJ34. The activation of FA/BRCA pathway was inhibited by downregulation of related factors including FANCD2, BRCA2 and Rad51. PJ34 significantly increased the ratio of γH2AX-positive cells and the number of foci/cells. The comet tail rate of cells, tail length, tail moment and Olive tail moment all increased after PJ34 treatment in RPMI8226/R cells. These results indicate that PJ34 combined treatment with melphalan produces synergistic effects and reverses multidrug resistance of RPMI8226/R cells effectively. PJ34 cannot induce DNA damage directly, but it may increase the DNA damage induced by melphalan through inhibiting DNA damage repair. The suppression of FA/BRCA pathway may be the mechanism. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of multidrug resistant multiple myeloma.

Topics: BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA Repair; Drug Resistance, Neoplasm; Drug Synergism; Fanconi Anemia Complementation Group A Protein; Humans; Melphalan; Multiple Myeloma; Phenanthrenes; Poly(ADP-ribose) Polymerase Inhibitors; Signal Transduction

As the principal active ingredient in the Chinese herb Tripterygium wilfordii Hook.F (TwHF), triptolide has been shown to have very strong antitumor properties. The trimethylation of lysine 4 on histone H3 (H3K4me3) has been proposed to promote gene expression, and the accumulation of H3K4me3 at the transcriptional start sites of oncogenes is involved in carcinogenesis. To identify the association between the reduction of H3K4me3 and the apoptosis of MM cells induced by triptolide, we investigated the global patterns of H3K4me3 occupancy in the MM cell genome. Combined analyses using ChIP-on-chip and western blotting showed that H3K4me3 were highly enriched on the gene promoters of c-Myc and VEGFA and were associated with the up-regulation of both genes. Treatment of KM3 cells with triptolide and siRNA targeting ASH2L reduced the expression of c-Myc and VEGFA. These results suggest that triptolide can down-regulate c-Myc and VEGFA expression by blocking the accumulation of H3K4me3 on their promoters,and thus play an important role in anti-MM mechanism.

Topics: Apoptosis; Cell Line, Tumor; Diterpenes; Epoxy Compounds; Histones; Humans; Methylation; Multiple Myeloma; Phenanthrenes

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.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; Epoxy Compounds; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Phenanthrenes; Transcription, Genetic

To investigate the effect of PARP1 inhibitor PJ34 on multi-drug resistance in a human multiple myeloma cell line and its connection with FA/BRCA pathway in DNA damage repair.. A CCK8 assay was used to measure the inhibition rate. Real-time quantitative PCR was used to detect expression changes of DNA repair genes involved in the FA/BRCA pathway. Western blotting assay was used to detect expression of key protein FANCD2 in the FA/BRCA pathway. Annexin VFITC/PI double staining flow cytometry was used to measure cell apoptosis induced by PJ34. A COMET assay was used to detect the effect of PJ34 on DNA damage repair.. PJ34 could significantly enhance the sensitivity of RPMI8226/R cells to melphalan. The IC50 value of melphalan was dropped from 20.43 mol/L to 7.8 mol/L. PJ34 could inhibit the DNA damage repair, and the effect was related with the inhibition of FA/BRCA pathway. PJ34 and melphalan showed a synergistic effect in promoting the apoptosis of RPMI8226/R cells.. PJ34 can reverse the resistance of RPMI8226/R cells to melphalan by inhibiting the FA/BRCA pathway, which in turn can induce suppression of DNA damage repair. Therefore, PJ34 may have clinical value in overcoming the multi-drug resistance of multiple myeloma.

Topics: Antineoplastic Agents; BRCA2 Protein; Cell Line, Tumor; Drug Resistance, Neoplasm; Fanconi Anemia Complementation Group D2 Protein; Humans; Multiple Myeloma; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases

Arsenic trioxide (As2O3) has been recently established as one of the most effective drugs for the treatment of patients with acute promyelocytic leukemia. However, it has exhibited to be less efficient for the non-promyelocytic leukaemia or other types of malignant tumors. The purpose of the present study was to explore new therapeutic strategies based on As2O3 for human multiple myeloma. Here, we first report cryptotanshinone (CPT) and As2O3 synergy for enhanced cytotoxicity in human multiple myeloma U266 cells. In particular, the apoptosis related proteins (e.g., cleaved poly (ADP-ribose) polymerase (PARP), caspase-3 and -9) were significantly increased by the combination treatment (iAs(III) + CPT), whereas, the expression of survival proteins such as Bcl-2 and survivin was suppressed, suggesting that the induction of apoptosis through mitochondrial-mediated apoptotic pathway. In addition, there were no appreciable effects observed in cells after exposure to either As2O3 or CPT alone. In order to better understand the molecular mechanism, we further determined the phosphorylation of STAT3, JNK, ERK and p38. Interestingly, phosphorylation of JNK and p38 were remarkably induced by combination treatment, and no significant inhibition of STAT3 or ERK was observed. In addition, induction of apoptosis in human multiple myeloma cells was partially abrogated only by pretreatment with JNK inhibitor and not by p38 inhibitor, suggesting that JNK pathway may play an important role in induction of apoptosis by the combination of iAs(III) and CPT. Further studies are needed to evaluate this synergistic anticancer effect in vivo. In the near future, this new approach might be used clinically for multiple myeloma (MM) treatment.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Blotting, Western; Cell Line, Tumor; Drug Synergism; Humans; Multiple Myeloma; Oxides; Phenanthrenes

The heat shock transcription factor 1 (HSF1) has recently been reported to promote malignant transformation and growth. Here we provide experimental evidence for a role of HSF1 in the pathogenesis of multiple myeloma (MM). Immunohistochemical analyses revealed that HSF1 was overexpressed in half of the investigated MM samples, including virtually all cases with extramedullary manifestations or anaplastic morphology. HSF1 function was inhibited either by siRNA-mediated knockdown or pharmacologically through treatment with triptolide. Both approaches caused depletion of HSF1, lowered the constitutively high expression of a multitude of protective HSPs (such as HSP90, HSP70, HSP40 and HSP27), induced apoptosis in human MM cells in vitro, and strongly reduced MM tumour growth in vivo. Furthermore, we observed that treatment-induced upregulation of HSPs after proteasome or HSP90 inhibition was critically dependent on HSF1. Importantly, the apoptotic effects of the HSP90 inhibitor NVP-AUY922 or the proteasome inhibitor bortezomib were strongly enhanced in combination with triptolide, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Collectively, our data indicate that inhibition of HSF1 affects multiple protective HSPs and might therefore represent a therapeutic strategy - in particular in combination with proteasome or HSP90 inhibitors.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cells, Cultured; Diterpenes; DNA-Binding Proteins; Down-Regulation; Epoxy Compounds; Female; Gene Knockdown Techniques; Heat Shock Transcription Factors; Heat-Shock Proteins; Humans; Immunohistochemistry; Isoxazoles; Mice; Mice, Inbred BALB C; Multiple Myeloma; Neoplasm Transplantation; Phenanthrenes; Plasma Cells; Proteasome Endopeptidase Complex; Pyrazines; Resorcinols; RNA, Small Interfering; Transcription Factors; Transplantation, Heterologous

To elucidate the relationship between triptolide-induced changes in histone methylation and its antitumor effect on human multiple myeloma (MM) cells in vitro.. Human multiple myeloma cell line RPMI8226 was used. Apoptosis was evaluated using Annexin-V-FITC/PI-labeled flow cytometry, Hoechst 33258 staining, and transmission electron microscopy. Flow cytometry was used to detect the cell cycle distribution of the apoptotic cells. The presence of the LSD1, JMJD2B, H3K4me2, H3K9me2, and H3K36me2 proteins was verified by Western blot analysis. Semi-quantitative real-time PCR was performed to examine the expression of LSD1 and JMJD2B.. Triptolide (10-160 nmol/L) suppressed the proliferation of MM cells in a dose- and time-dependent manner with an IC(50) value of 99.2 ± 9.0 nmol/L at 24 h. Triptolide (50 nmol/L) induced G(0)/G(1) cell cycle arrest in MM cells. The agent (50-150 nmol/L) induced apoptosis of MM cells in a dose-dependent manner. The same concentrations of triptolide suppressed the expression of dimethylated H3K4, dimethylated H3K9 and dimethylated H3K36 by altering the expression of histone demethylase LSD1 and JMJD2B without affecting the expression of histone demethylase LSD1.. Triptolide potently inhibits the growth of MM cells via regulating the expression of histone demethylase LSD1 and JMJD2B, which lead to abnormal histone methylation.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Diterpenes; DNA Methylation; Epoxy Compounds; Histone Demethylases; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Lysine; Multiple Myeloma; Phenanthrenes

Recently triptolide (TPL) has been proved to have the capacity to inhibit the proliferation of multiple myeloma (MM) cells as well as leukemic cells in vitro. In the present study, we found a synergistic effect when TPL was added to dexamethasone to induce apoptosis in MM.1S cells. This combination induced a significantly higher proportion of apoptotic cells compared with those treated with each drug separately. TPL down-regulated the expression of miR142 - 5p and miR181a, which have been shown to inhibit glucocorticoid receptor (GR) expression. MicroRNA mimics and inhibitors inhibited or enhanced the synergistic effect between TPL and dexamethasone in inducing apoptosis in MM.1S cells, suggesting an important role of miR142 - 5p and miR181a in GR regulation by TPL. The in vitro proapoptotic effect of TPL associated with dexamethasone reveals a new lead for further clinical investigation into the treatment of patients with MM with TPL.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dexamethasone; Diterpenes; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Epoxy Compounds; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Multiple Myeloma; Phenanthrenes

This study was purposed to investigate the effect of triptolide on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929(H929). MTT assay was applied to detect the inhibitory effects of triptolide and bortezomib alone or combined at different concentrations on H929 cells, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining. The results showed that both triptolide (10 - 100 ng/ml) and bortezomib (10 - 100 nmol/L) alone or combination inhibited the proliferation of MM cell line H929 in a concentration-dependent manner. The apoptotic rate of H929 cells in group of triptolide combined with bortezomib was much higher than that in groups of single drug or control; moreover, the apoptotic rate of H929 cells treated by non-inhibitory concentration of triptolide (10 ng/ml) combined with bortezomib (40 nmol/L) for 24 h was significantly higher than that by bortezomib alone (P < 0.05). It is concluded that triptolide can significantly enhance the pro-apoptotic activity of bortezomib in MM cells.

Topics: Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Diterpenes; Epoxy Compounds; Humans; Multiple Myeloma; Phenanthrenes; Pyrazines

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro, as well as the roles of nuclear factor-kappa B (NF-κB) and IκBα was investigated. The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay. Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay. The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy. The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells. Triptolide at nM range induced proliferation inhibition in a dose- and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells. Besides, we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol. These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.

Topics: Apoptosis; Cell Line, Tumor; Diterpenes; Epoxy Compounds; Humans; Multiple Myeloma; NF-kappa B; Phenanthrenes; Signal Transduction

Triptolide is the principal active ingredient in extracts from the Chinese herb Tripterygium wilfordii Hook.F (TwHF), and has various functions such as immunosuppression, anti-inflammatory and antitumor properties. In diverse hematological tumors triptolide exerts antitumor activity and many studies have tried to elucidate the potential antitumor mechanism. The evidence that triptolide-induced gene promoter DNA hypermethylation has suggested that epigenetic mechanisms may play an important role in the antitumor activity of triptolide. Our study aimed to investigate the association of the therapeutic effect of triptolide on multiple myeloma with the regulation of histone methylation. Triptolide inhibited the proliferation of multiple myeloma cell line RPMI8226 in a time- and dose-dependent manner, induced G0/G1 cell cycle arrest and apoptosis. Triptolide decreased histone H3K9 and H3K27 methylation via the downregulation of histone methyltransferase SUV39H1 and EZH2, respectively, which possibly was the anti-myeloma mechanism of triptolide.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Diterpenes; Epoxy Compounds; G1 Phase; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; Humans; Interphase; Methylation; Multiple Myeloma; Phenanthrenes; Resting Phase, Cell Cycle

To investigate the effects of triptolide on proliferation and apoptosis as well as on the expression of RIZ1 in the human multiple myeloma cell line U266 in vitro.. The effect of triptolide on the growth of U266 cells was studied by MTT assay. Apoptosis was detected by Hoechst 33258 staining and Annexin V/PI double-labeled flow cytometry, and caspase-3 mRNA was measured by RT-PCR. Western blotting, flow cytometry and RT-PCR were used to assess the expression of RIZ1, and the location and expression of H3K9me1 were detected by confocal microscopy and Western blotting.. Triptolide significantly inhibited the proliferation of U266 cells in a time- and concentration-dependent manner (the IC(50) value for a 24-h exposure was 157.19+/-0.38 nmol/L). Triptolide induced typical apoptotic morphological changes. Triptolide 40, 80, and 160 nmol/L treatment induced significant caspase-3-dependent apoptosis compared with control group (10.5%+/-1.23%, 37.9%+/-2.45%, and 40.5%+/-2.30% vs 3.8%+/-1.98%, P<0.05). Compared with peripheral blood monocular cells (PBMC) from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, but the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. Triptolide increased the protein expression of RIZ1 and RIZ1 methylates histone H3 lysine 9 in U266 cells.. Triptolide increased the protein expression of RIZ1, inhibited the proliferation, and induced caspase-dependent apoptosis in U266 cells.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Bisbenzimidazole; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Diterpenes; DNA-Binding Proteins; Drugs, Chinese Herbal; Epoxy Compounds; Flow Cytometry; Fluorescent Dyes; Histone-Lysine N-Methyltransferase; Histones; Humans; Inhibitor of Apoptosis Proteins; Methylation; Multiple Myeloma; Nuclear Proteins; Phenanthrenes; Transcription Factors

Multiple myeloma is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in multiple myeloma. Histone lysine methylation has emerged as a central epigenetic change in the organization of eukaryotic chromatin with far-reaching implications for the regulation of cell proliferation, cell-type differentiation, gene expression, genome stability, overall development, and genesis of cancer. Triptolide is the principal active ingredient in extracts from the Chinese herb Tripterygium wilfordii Hook.F (TwHF), and numerous studies have elucidated its antitumor property. Our experiments discovered that triptolide inhibited the proliferation of multiple myeloma cell line U266 in a time- and dose-dependent manner, induced G2/M cell cycle arrest and caspase-dependent apoptosis. Triptolide could decrease the expression of histone H3K4, H3K27 and H3K36 trimethylation in parallel with histone methyltransferases SMYD3, EZH2 and NSD1 respectively, which possibly was the anti-myeloma mechanism of triptolide.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Diterpenes; Epoxy Compounds; G2 Phase; Gene Expression Regulation, Neoplastic; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; Humans; Methylation; Multiple Myeloma; Phenanthrenes; RNA, Messenger

Glucocorticoids are widely used chemotherapeutic agents for multiple myeloma. Drug resistance to steroid therapies is associated with the downregulation or loss of glucocorticoid receptor expression in malignant plasma cells. In this study, we examined the constitutive expression of glucocorticoid receptor in dexamethasone-sensitive and dexamethasone-resistant multiple myeloma cell lines. We found that triptolide increased the amount of the phosphorylated glucocorticoid receptor and enhanced the growth inhibitory effect of dexamethasone. Notably, these effects could not be blocked by interleukin-6, one of the most important growth factors in multiple myeloma.

Topics: Antineoplastic Agents, Alkylating; Cell Line, Tumor; Dexamethasone; Diterpenes; Drug Resistance, Neoplasm; Epoxy Compounds; Humans; Interleukin-6; Multiple Myeloma; Phenanthrenes; Phosphorylation; Receptors, Glucocorticoid; Up-Regulation

Human multiple myeloma is a presently incurable hematological malignancy and novel biologically based therapies are urgently needed. Triptolide (TPL) is a purified diterpenod isolated from the Chinese herb, Tripterygium wilfordii Hook. F that has shown antitumor activities in various cancer cell types. But its activity in Dex-resistant multiple myeloma cell lines and the main upstream signaling pathway has not been reported. Here we show that TPL induces apoptosis in dexamethasone-sensitive (MM.1S) and dexamethasone-resistant (MM.1R) cells, most importantly its main upstream signaling pathway is through the PI3k/Akt/NF-kappaB pathway and is also associated with MAPK pathway, via mitochondrial apoptotic signaling and is also associated with the caspase and Bcl-2 family members. Moreover, TPL was able to enhance the activities of dexamethasone or bortezomib/PS-341 in multiple myeloma cell lines. Collectively, these findings provide the framework for a clinical evaluation of TPL, either alone or in combination with dexamethasone or bortezomib/PS-341, to overcome drug resistance and improve outcome for patients with this universally fatal hematological malignancy.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Dexamethasone; Diterpenes; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Epoxy Compounds; Humans; I-kappa B Proteins; Inhibitor of Apoptosis Proteins; Mitochondria; Multiple Myeloma; NF-kappa B; Phenanthrenes; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Receptors, Glucocorticoid; Up-Regulation

Triptolide has been reported to be effective in the treatment of auto-immune diseases. This study investigates the cytotoxic function of triptolide on multiple myeloma (MM) cells. We found that triptolide inhibited the proliferation of both RPMI8226 and U266 cells in a dose-dependent manner (10-80 ng/mL). Triptolide induced apoptosis in MM cells through activation of the cystein protease caspase 8, 9 and 3, and subsequent cleavage of the DNA repair enzyme poly (ADP-ribose) polymerase. Apoptosis was confirmed with cell-cycle analysis and annexin V staining. Moreover, triptolide down-regulated nuclear factor (NF)-kappaB activity in MM cell lines. In addition, triptolide also induced chemosensitivity to doxorubicin and suppressed cell proliferation of fresh MM cells. Therefore, triptolide appears to be a potent inducer of apoptosis in myeloma cells, and might have some benefit in the treatment of myeloma patients.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Caspase 8; Caspase 9; Caspases; Cell Proliferation; Diterpenes; Enzyme Activation; Epoxy Compounds; Humans; Multiple Myeloma; NF-kappa B; Phenanthrenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Transcription Factors; Tumor Cells, Cultured