*Alkaloids ; *Antineoplastic Agents, Phytogenic ; Chemistry ; *Harringtonines

Alkaloids ; Antineoplastic Agents, Phytogenic ; Chemical Phenomena ; Chemistry ; Harringtonines

Alkaloids ; Apoptosis ; HL-60 Cells ; Harringtonines ; Humans ; Quinolizines

The study was aimed to investigate the synergistically effect of interferon-α (IFN-α) and homoharringtonine (HHT) on the proliferation, apoptosis, cell cycle of K562 cells and the expression of β-catenin. The proliferation, apoptosis, cell cycle and β-catenin mRNA expression of K562 cells treated with IFN-α and/or HHT were assayed with MTT, flow cytometry or RT-PCR respectively. The results showed that HHT alone, but not IFN-α alone, displayed a proliferation inhibition, apoptosis induction, G(0)/G(1) phase block and down-regulation of β-catenin expression in K562 cells with concentration- and time-dependent manners. The expression level of β-catenin mRNA after being treated with HHT was 0.5576 ± 0.0373, which were lower than that in control group (0.9369 ± 0.0142). The down-regulation of β-catenin expression in group of IFN-α combined with HHT was higher significantly than that in HHT group (0.3737 ± 0.0529 vs 0.5576 ± 0.0373, P < 0.05). Otherwise, HHT combined with IFN-α did not demonstrate obvious toxicologic effect on bone marrow mononuclear cells. It is concluded that IFN-α combined with HHT can enhance the cytotoxic effect of HHT on K562 cells, which may be associated with down-regulation of β-catenin expression.
Cell Proliferation ; drug effects ; Harringtonines ; pharmacology ; Humans ; Interferon-alpha ; pharmacology ; K562 Cells ; beta Catenin ; genetics ; metabolism

OBJECTIVETo explore the effects of BTZ plus HHT on proliferation and apoptosis of K562 cells, and to clarify the relationship between the mechanism inderlying the effect of BTZ plus HHT on K562 cells and BCL-2, BAX, MCL-1 proteins.

METHODSThe K562 cells were divided into 4 groups by different treatment: BTZ(20 nmol/L), HHT(40 ng/ml), BTZ(20 nmol/L)+HHT(40 ng/ml) and control. The proliferation inhibition rates of K562 cells in each group were detected by using MTT, and the early apoptosis rates of K562 cells in each group were assayed by using flow cytometry with Annexin V-FITC/PI staining. The proteins level of BCL-2, BAX and MCL-1 in each group were examined by using Western blot.

RESULTSThe inhibition rate of K562 cell proliferation in combined group was higher than that in BTZ, HHT alone group(P<0.01). The early apoptosis rate of K562 cells in combined group was increased significantly in comparison with BTZ and HHT alone group(P<0.05). The BCL-2 protein level of K562 cells in combined group was significantly lower than that in BTZ and HHT alone group(P<0.05). BAX protein level of K562 cells in combined group was higher than that in BTZ and HHT alone group(P<0.05). The Orders of the MCL-1 protein level of K562 cells in 4 groups were BTZ>Control>BTZ plus HHT>HHT(P<0.05 ).

CONCLUSIONThe combination of BTZ and HHT exerts the synergistic effect of anti-proliferative activity and induces apoptosis against K562 cells in vitro. The combination can induce apoptosis of K562 cells via suppression of BCL-2 protein and up-regulation of BAX protein. HHT can increase the sensitivity of K562 cells to BTZ by down-regulating the expression of MCL-1 protein.

Apoptosis ; Bortezomib ; pharmacology ; Cell Proliferation ; Harringtonines ; pharmacology ; Homoharringtonine ; Humans ; K562 Cells

OBJECTIVETo investigate the effect and possible mechanism of low concentration of triptolide (TPL) combined with homoharringtonine (HHT) on the proliferation and apoptosis of KG-1α cells.

METHODSCCK-8 method was used to detect the antiproliferating effects of different concentrations of TPL and HHT single-use and combined use on KG-1α cells, and the combined index (CI) was calculated. The colony formation ability was also determined by methylcellulose colony formation assay, cell surface molecules, apoptosis rate and cell cycle changes were detected by flow cytometry. Westerrn blot was used to detect the expression of Akt signaling pathway related proteins before and after low dose TPL combined with HHT using.

RESULTSHigh expression of CD34 and CD123 were on KG-1a cells, which being lack expression of CD38. TPL and HHT dose-dependently inhibited the proliferation of KG-1α cells. Compared with low dosage TPL and HHT single-use groups, the cell proliferation and colony formation efficiency were lower, and the cell apoptosis rate was higher in the combined group. CI values also indicated that low concentration TPL combined with HHT possessed highly synergistic effect. After the combination of the 2 drugs, the expressions of P-Akt, P-Akt, BCL-2, PARP and survivin protein were down-regulated and the cleavage of PARP protein was increased.

CONCLUSIONLow concentration of TPL combined with HHT can synergistically inhibit KG-1α cell proliferation and induce its apoptosis through the PI3K/Akt signaling pathway and downstream protein.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Diterpenes ; Epoxy Compounds ; Harringtonines ; Homoharringtonine ; Humans ; Phenanthrenes ; Phosphatidylinositol 3-Kinases

OBJECTIVETo establish a homoharringtonine (HHT)-resistant SKM-1 cell line and explore its biologic characteristics and mechanisms for drug resistance.

METHODSThe HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level. The morphology of the resistant and parental cell lines was observed through optical microscope. The MTT assay was used to determine the doubling time and the resistance index to draw growth curve. The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry, and the karyotypes by R-banding. Semi-quantitative real-time PCR was performed to evaluate the expression levels of mdr1, MRP and topo-IIa.

RESULTSThe HHT-resistant cell line SKM-1/HHT was eventually established following 7-month drug induction. Both the resistant and the parental cell lines were similar with regard to morphology and immunophenotype. The karyotypes of the former was more complicated with differences located in chromosome 20, X, 4, 5, 9 and 11. The resistant cell line had more G(1) phase cells (64.04% vs 41.91%), less S phase cells (34.92% vs 53.53%), and less G(2) phase cells (1.04% vs 4.56%) compared with the parental cell line. The SKM-1/HHT cell line showed significant drug resistance to HHT, VCR, DNR and etoposide, the resistance indices of HHT, VCR, DNR and etoposide were 17.94, 8.75, 5.99 and 13.76 respectively. DNR accumulation was impaired in SKM-1/HHT cell line as less fluorescence of DNR (698 ± 36 vs 858 ± 54). The expression of mdr1 increased dramatically in the resistant cell line, its 2(-ΔCt) value was 20.1 higher than that of the parental cell line \[(3.42 ± 0.46)×10(-2) vs (0.17 ± 0.01)×10(-2), P < 0.05\], while MRP also increased in the resistant by 3.56 folds \[(4.77 ± 0.87)×10(-3) vs (1.34 ± 0.56)×10(-3), P < 0.05\]; However there was a slightly decrease of topo-IIa, the ratio of the resistant to the parental calculated by their 2(-ΔCt) values was 0.619:1 \[(1.91 ± 0.30)×10(-4) vs (3.08 ± 0.21)×10(-4), P < 0.05\].

CONCLUSIONA HHT-resistant cell line SKM-1/HHT was established. The prominent overexpression of mdr1 may be the main cause for multidrug resistance.

Cell Line, Tumor ; Drug Resistance, Neoplasm ; Harringtonines ; pharmacology ; Humans ; Leukemia, Myeloid, Acute ; pathology ; Myelodysplastic Syndromes ; metabolism ; pathology

This study was aimed to explore the effect of homoharringtonine in combination with AG490 on JAK2-STAT5 associated signal pathway in HEL cells, and analyze its mechanism so as to provide theoretical basis for therapy of chronic myeloproliferative neoplasma by new program. The cell survival rates were tested by MTT, apoptosis was tested by flow cytometry after HEL cells were treated by 20 ng/ml HHT, 100 µmol/L AG490 and 20 ng/ml HHT in combination with 100 µmol/L AG490, while the signal proteins such as P-JAK2, P-STAT5 and BCL-xL activated by abnormal activated JAK2 were tested by Western blot. The results showed that both HHT and AG490 could inhabit the HEL cell proliferation after being treated for 24 hours, and Annexin V-PI double staining confirmed early apoptosis while HHT effect was more obvious, Western blot showed that the expressions of P-JAK2 and P-STAT5 were down-regulated, while the total protein levels of JAK2 and STAT5 were stable. It is concluded that HHT combined with AG490 can obviously inhibit the proliferation and induce early apoptosis of HEL cells, and there is synergistic effect between the two drugs. HHT possibly acts as a broad-spectrum PTK inhibitor and synergistically with AG490 inhibits the phosphorylation of signal proteins caused by JAK2V617F, thus down-regulating the transcription of STAT5.
Cell Line, Tumor ; Harringtonines ; pharmacology ; Humans ; Janus Kinase 2 ; metabolism ; STAT5 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tyrphostins ; pharmacology

This study was aimed to investigate the effect of homoharringtonine (HHT) on K562 cell proliferation, apoptosis and expression of BCL-2 and NF-κB proteins. The cells proliferation was assayed with MTT method, the cell apoptosis, cell cycle and BCL-2 expression were analyzed with flow cytometry, NF-κB protein expression was detected with Western blot. The results showed that HHT concentration-dependently inhibited proliferation of K562 cells, the IC50 at 48 h was 43.89 ng/ml. Treated with HHT 10 ng/ml for 48 h, K562 cell apoptosis significantly increased, cell cycle was blocked at G0/G1, the expression level of BCL-2 and NF-κB proteins was lower than that in control group (P < 0.05). It is concluded that HHT may inhibit the proliferation of K562 cells, and down-regulating expression levels of BCL-2 and NF-κB may be one of its anti-CML mechanisms.
Flow Cytometry ; Harringtonines ; pharmacology ; Humans ; K562 Cells ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo screen and identify apoptosis related proteins and explore the mechanism of harringtonine (HT)-induced K562 cells apoptosis.

METHODSFlow cytometry was used to distinguish K562 cells in the earlier stage of apoptosis from those in the later stage of apoptosis by annexin V and PI staining. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with computer image analysis was used to detect the changes in protein expression in the two stages of apoptosis. Proteins were identified by peptide mass fingerprint in combination with database searching.

RESULTSK562 cells treated with HT for 5 and 24 hours were in the early and later stages of apoptosis respectively. Statistical analysis showed 3 spots disappeared, 7 spots with decreased intensity and 10 spots with increased intensity in the 24 h HT induced apoptotic cells as compared with that in 5 h HT induced ones. Ten spots were selected on the basis of the intensity and the significant changes in abundance. Among them, 5 apoptosis related proteins were successfully identified by MALDI-TOF: keratin 9, BTF3, TrpRS, RS and prohibitin.

CONCLUSIONSUp-regulation of TrpRS, RS, prohibitin and down-regulation of BTF3 were involved in inhibition of transcription and protein synthesis in the apoptotic K562 cells induced by HT, whereas up-regulation of keratin 9 was related to apoptosis resistance.

Apoptosis ; drug effects ; Electrophoresis, Gel, Two-Dimensional ; Harringtonines ; pharmacology ; Humans ; K562 Cells ; drug effects ; metabolism ; pathology ; Proteome ; metabolism