OBJECTIVETo investigate the effect of bortezomib (Bor) alone or in combination with As(2)O(3) (ATO) and/or dexamethasone (DXM) on proliferation and apoptosis in KM3 human multiple myeloma cell line KM3.

METHODSKM3 cells were cultured with different concentrations of Bor and ATO and/or DXM in combination or Bor, ATO, DXM alone for different times. Cell proliferation was assayed by MTT assay, and IC(50) was calculated. Cell morphology was observed with light and electric microscopy. The agarose gel electrophoresis was used to evaluate DNA content, and the flow cytometry was used to exam Annexin V-FITC/PI stain.

RESULTSBor, ATO and DXM inhibited KM3 cell proliferation in a time-and dose-dependent manner with the IC(50) of 0.27, 3.10 and 8.01 micromol/L, respectively. The inhibition rate of KM3 cells by Bor plus ATO and DXM was significantly higher than Bor plus ATO or DXM \[(34.51 +/- 0.51)% vs (25.39 +/- 0.90)% and (34.51 +/- 0.51)% vs (23.80 +/- 0.78)% respectively\]. Typical morphology for apoptosis and DNA ladder were observed in KM3 cell treated with 0.25 micromol/L Bor for 48 h, by Annexin V positivity. The apoptosis rate induced by Bor plus both ATO and DXM was higher than that induced by Bor plus DXM.

CONCLUSIONBor can inhibit the proliferation and induce apoptosis of KM3 cells. Bor enhances the inhibitory effect of ATO and DXM on the growth of KM3 cell. ATO enhances the apoptosis effects of Bor and DXM on KM3 cells.

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Humans ; Multiple Myeloma ; metabolism

OBJECTIVETo investigate the effect of bortezomib (Bor) alone and in combination with arabinoside (Ara-C) on proliferation and apoptosis of leukemia cell line K562.

METHODSK562 cells were treated with 20 nmol/L Bor and 0.2 microg/ml Ara-C alone and in combination for 48 h. MTT was used to study the inhibitory effects on cell growth and the apoptosis rate was analysed by flow cytometry. After K562 cells treated with 20 nmol/L Bor or 0.2 microg/ml Ara-C for 6 h, the activity of NF-kappaB was analyzed by SP immunohistochemistry and cell cycle by flow cytometry.

RESULTSThe inhibition and apoptosis rates of K562 cells in combination groups were higher than those in the two single treatment groups (P < 0.01), especially in the combined treatment group in which K562 cells were treated first with Ara-C for 6 h then with Bor combined,the inhibition and apoptosis rates were the highest [(81.5 +/- 4.0)% and (29.2 +/- 3.1)%, respectively] (P < 0.01). In the other two combined groups in which the cells were treated with Bor for 6 h then with Ara-C combined, or treated with the two drugs simultaneously, the inhibition and apoptosis rates were (54.1 +/- 4.2)% and (18.7 +/- 3.5)%, and (66.2 +/- 2.8)% and (21.1 +/- 2.2)%, respectively. Treatment of K562 cells with 20 nmol/L Bor for 6 h, the activity of NF-kappaB was decreased significantly, and the cells were apparently arrested in G(2)/M phase, and treatment with 0.2 microg/ml Ara-C in the same manner, the activity of NF-kappaB was increased significantly, and the cells were apparently arrested in G(1) phase.

CONCLUSIONSBor can effectively inhibit K562 cell proliferation, and induced its apoptosis. This effect was enhanced significantly when in combination with Ara-C. Pretreatment of K562 cells with Ara-C lead to the increased activity of NF-kappaB and the fraction of G(1) phase cells.

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Proliferation ; drug effects ; Humans ; K562 Cells ; Pyrazines ; pharmacology

OBJECTIVETo investigate the synergistic anti-multiple myeloma (MM) effect of 2-methoxyestradiol (2-ME2) and bortezomib, and explore the relationship between this effect and blockade of aggresomes formation by 2-ME2.

METHODSFour MM cell lines RPMI-8226, NCI-H929, U266 and SKO-007 were used for study. Immunoflourescent anti-ubiquitin and Hoechst 33342 staining were used to examine aggresome-positive cells and apoptotic cells, respectively. Isobolographic analysis was used for determination of synergy.

RESULTS(1) Quantitative assay showed that in the absence of bortezomib, only 6.6% - 8.9% of MM cells were aggresome-positive, but the percentage was increased to 71.9%-83.4% after treatment with bortezomib at IC(20) concentration for 24 h. Aggresome-positive cells with immunoreactivity to anti-ubiquitin were detected in almost all non-apoptotic cells, but not in apoptotic cells. (2) Treatment in a definite range of concentrations bortezomib plus 2-ME2 led to MM cell apoptosis compared with each agent alone and the significantly synergistic effect confirmed by isobolographic analysis. (3) Combination of bortezomib and 2-ME2 increased the apoptotic cells aggresome-negative cells (ANK) and decreased the non-apoptotic cells in aggresome positive cells (APC). In RPMI8226 and U266 cells, the apoptotic cells in ANC increased from (14.5 +/- 2.0)% and (20.1 +/- 2.9)% to (80.7 +/- 6.9)% and (71.6 +/- 6.2)%, and the non-apoptotic cells in APC decreased from (75.3 +/- 5.7)% and (69.1 +/- 8.6)% to (13.8 +/- 3.8)% and (19.5 +/- 4.2)%, respectively, in combined group and bortezomib alone group.

CONCLUSIONBortezomib-induced aggresomes have a protective function for MM cells and combination of bortezomib with 2-ME2 induced a synergistic cytotoxicity to the cells.

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Humans ; Multiple Myeloma ; metabolism ; Pyrazines ; pharmacology

This study was aimed to explore the effect of bortezomib and low concentration cytarabine (Ara-C) on proliferation and apoptosis in U937 cell line and its mechanism. The proliferation and apoptosis of U937 cells treated with bortezomib (10 nmol/L) and(or) Ara-C (50 nmol/L) were observed by cell count, cell morphology, flow cytometry and Western blot. The results showed that bortezomib and Ara-C alone inhibited U937 cell proliferation. The inhibitory effect was enhanced by combination of these two drugs, the inhibitory rates of U937 cell proliferation were (55.00 ± 2.81)% and (70.02 ± 3.33)% after treatment for 24 h and 48 h, respectively. Bortezomib and Ara-C synergistically induced apoptosis and decreased mitochondrial membrane potential in U937 cells. The percentage of Rhodamin123 positive cells was (38.70 ± 1.54)%. Bortezomib and Ara-C also synergistically induced activation of caspase-9, caspase-8 and caspase-3. It is concluded that the bortezomib and low concentration Ara-C synergistically induced apoptosis in U937 cells, mainly through mitochondrial pathway, and possibly through death receptor pathway.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Cycle ; drug effects ; Cytarabine ; pharmacology ; Humans ; Pyrazines ; pharmacology ; U937 Cells

Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Hepatic Stellate Cells ; cytology ; drug effects ; Protease Inhibitors ; pharmacology ; Pyrazines ; pharmacology

The purpose of this study was to explore the effect of proteasome inhibitor, bortezomib (Bzb), on osteoblast in pathologic status of myeloma bone disease. The myeloma bone disease was modeled by co-culture of mouse myeloma cell RPMI8226 with osteoblast line MC-3T3E1 from mouse calvaria, and intervenient culture of supernatant. The inhibitory effect of Bzb on proliferation of MC-3T3E1 assayed by modified MTT method, the apoptosis of MC-3T3E1 cells was determined by flow cytometry with Annexin V/PI staining, the expressions of osteoblast markers, Runx2/cbfa1, osteocalcin (OCN) and osterix (OSX) in MC-3T3E1 treated with Bzb were detected by RT-PCR and Western blot respectively. Experiments were divided into 3 group: single cultured, co-cultured and supernatant-interveniently cultured groups. The results showed the Bzb in higher concentration inhibited proliferation of MC-3T3E1 cells in a dose-dependent manner, with the IC(50) of 38.1 nmol/L for 48 hours, the Bzb in low concentration (5 nmol/L) did not show the inhibitory effect on proliferation of MC-3T3E1 in single cultured group (p>0.10), but could decrease apoptotic rate of MC-3T3E1 by 32.5% and 24.6% respectively in cocultured and supernatant-interveniently cultured groups, moreover increased the expression of osteoblast-related gene OSX, OCN mRNA and protein (p<0.05), while no obvious change of Runx2/cbfa1 expression was observed (p>0.05). It is concluded that the proteasome inhibitor, Bzb, in low concentration promotes the activity of osteoblast internal mechanisms, and prevents the apoptosis of osteoblasts induced by myeloma cells. In addition, it can up-regulate transcription and expression of osteoblast markers related to Runx2/cbfa1 path way, thus may protect osteoblasts in myeloma bone disease.
3T3 Cells ; Animals ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Mice ; Multiple Myeloma ; pathology ; Pyrazines ; pharmacology

The aim of this study was to explore the synergistic effect of arsenic trioxide and bortezomib on apoptosis of Raji cell line. The cells were treated with arsenic trioxide, bortezomib, low-dose arsenic trioxide combined with bortezomib, respectively. The cell viability and proliferative curve were estimated by trypan blue dye exclusion. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The apoptosis related elements such as caspase-3, BCL-2, BAX, JNK2 and IkappaB-alpha, were measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.01), and apoptosis rate markedly increased (p = 0.001), while obvious cell cycle arrest was not observed. On the protein level, the expression of Caspase-3, BAX and IkappaB-alpha increased, while the expression of BCL-2, and JNK2 decreased. It is concluded that low-dose arsenic trioxide combined with bortezomib synergistically induced apoptosis in Raji cell line which may be mediated by inhibiting NK-kappaB and JNK2 signaling.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Boronic Acids ; pharmacology ; Bortezomib ; Burkitt Lymphoma ; pathology ; Cell Line, Tumor ; Drug Synergism ; Humans ; Oxides ; pharmacology ; Protease Inhibitors ; pharmacology ; Pyrazines ; pharmacology

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.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Humans ; Multiple Myeloma ; pathology ; Phenanthrenes ; pharmacology ; Pyrazines ; pharmacology

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Oxides ; pharmacology ; Pyrazines ; pharmacology

This study was aimed to investigate the effect of arsenic trioxide (As(2)O(3)) alone and in combination with bortezomib (Bor) on proliferation and apoptosis of leukemia cell line K562, and to analyze the potential mechanism. K562 cells were treated with different concentrations of As(2)O(3) or Bor (alone or combination) for 24, 48 h. MTT method was used to detect the cell proliferation. After K562 cells were treated with 0.5 µmol/L As(2)O(3) alone or in combination with 10 nmoL/L Bor, the apoptosis rate and cell cycle were measured by flow cytometry, and the activity of NF-κB was analyzed by SP immunohistochemistry. The results indicated that the different concentrations of As(2)O(3) and Bor could inhibit the K562 cell proliferation in a time- and dose-dependent manners (P < 0.05). The IC(50) of Bor and As(2)O(3) in 48 h were 20 nmol/L and 0.6 µmol/L respectively. When K562 cells were treated with As(2)O(3) or Bor alone for 24 h, the apoptotic rate of K562 cells increased, and the apoptotic rate in combination group was higher than that in As(2)O(3) or Bor group. The cells were apparently arrested in G(2)/M phase in Bor group and G(0)/G(1) phase in As(2)O(3) group. The activity of NF-κB decreased significantly in As(2)O(3) or Bor group (P < 0.05), this effect was most significant in the combination group (P < 0.01). It is concluded that both As(2)O(3) and Bor can inhibit the proliferation and induce apoptosis of K562 cells, a synergistic effect can be observed when a low dose of As(2)O(3) combined with low dose of Bor. The different cell cycle block site and the decrease of activity of NF-κB may be one of the mechanisms underlying their synergic effect.
Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Proliferation ; drug effects ; Drug Synergism ; Humans ; K562 Cells ; NF-kappa B ; metabolism ; Oxides ; pharmacology ; Pyrazines ; pharmacology