| Gossypol, a natural polyphenol extracted from cottonseed, has been shown to have antiproliferative activity in a variety of cancer cell lines. (-)gossypol was proved the potent component and regarded as a small-molecule inhibitor of antiapoptosis protein Bcl-2 and Bcl-XL proteins.Chronic myeloid leukemia (CML) is a hematopoietic neoplasm with the hallmark of chimeric oncoprotein, Bcr-Abl, which displays constitutive tyrosine kinase activity and activates several signaling transduction pathways. Imatinib (Gleevec, STI-571; Novartis, Inc.) is a tyrosine kinase inhibitor that competitively inhibits Bcr-Abl kinases. It specifically induces apoptosis both in vitro and in vivo, Imatinib has shown a substantial clinical effect in BCR/ABL-positive leukemia patients. Although demonstrating impressive clinical activity against chronic-phase CML, in the accelerated and blastic phases of CML (CML-BC) the outcome after imatinib therapy is unacceptably poor and imatinib resistance occur shortly after the treatment. These findings emphasize the need to identify novel therapies for CML.In the present studies, we discussed the combination effect of (-)gossypol and imatinib on CML cell line K562, along with the effects of (-)gossypol on K562 and K562/R.1. Inhibition effects of (-)gossypol and its combination with imatinib on K562 cells.Effects of (-)gossypol and the combination of (-)gossypol with imatinib on the growth of K562 cells were analyzed by MTT assay. The growth of K562 cells was inhibited by (-)gossypol with the IC50 value of 5.22μmol/L. At the concentration of 6.25μmol/L (-)gossypol reduced the IC50 value of imatinib from 1.56μmol/L to 0.013μmol/L. In vivo result in nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562 cells in nude mice, and co-treatment with (-)gossypol 15mg/kg and Imatinib 30μmol/kg inhibited K562 cell growth by 93.7%. At a fixed ratio of 10:1, combination index value of (-)gossypol and imatinib < 1.0, corresponding to synergistic interactions, was obtained in MTT assay.Morphologically, combination of (-)gossypol 5μmol/L with imatinib 0.5μmol/L dramatically induced typical apoptotic change -- karyorrhexis in K562 cells, whereas the effect of either (-)gossypol 5μmol/L or imatinib 0.5μmol/L was barely seen. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis, by staining with PI as well as Annexin V/PI, similar results came out that percentage of apoptotic K562 cells increased markedly after combination treatment with (-)gossypol and imatinib. PI staining showed that (-)gossypol increased apoptosis percentage and caused G0/G1 arrest of cell cycle in K562 cells in a dose-dependent manner. By using DiOC6, loss ofΔΨm was also detected in (-)gossypol and imatinib treated K562 cells, which represent mitochondria function. Combination of (-)gossypol 5μmol/L and imatinib 0.5μmol/L leaded toΔΨm loss to a deeper extent in K562 cells .In order to understand the pathways involved in (-)gossypol and imatinib induced apoptosis, the level of caspase3 and cytoplasmic cytochrome C were tested using western blot. Procaspase3 decreased and cytoplasmic cytochrome C increased after treatment with (-)gossypol or imatinib. Combination with (-)gossypol and imatinib increased cytoplasmic cytochrome C more, and decreased procaspase3 with cleaved fragments of caspase3 detected, which it predicted the activation of caspase3. It can be inferred that treatment with (-)gossypol and imatinib arose cytochrome C release in K562 cells and consequently activated caspase3. To highlight the mechanism of growth inhibition and apoptosis induced by (-)gossypol and imatinib, protein and mRNA expression of several Bcl-2 family members and P21 were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-XL, Mcl-1 protein level moderately and Bcl-2 protein level neglectablely. Combination with (-)gossypol and imatinib caused Bcl-XL, Mcl-1 protein level to decrease markedly and had no obvious effect on Bcl-2 protein level. As far as the mRNA level was concerned, combination with (-)gossypol and imatinib decreased level of Bcl-2, Bcl-XL, Mcl-1, Bak, and increased level of Bad, whereas (-)gossypol simply decreased Bcl-XL level. P21 induction was observed in (-)gossypol treated K562 cells, however, imatinib seemed to have reverse effect on P21 induction. Combination with (-)gossypol and imatinib reduced expression of Bcr-Abl and JNK, while neither (-)gossypol nor imatinib caused detectable change of these two proteins at tested concentrations.Conclusively, (-)gossypol inhibited the growth of CML cell line K562 by inducing apoptosis and G0/G1 arrest in cell cycle, (-)gossypol synergistically potentiated imatinib induced apoptosis in K562 cells. Mictochondria pathway contributed at least partly to (-)gossypol induced apoptosis with the decreased Bcl-XL, Mcl-1 expression. Although (-)gossypol did not effect Bcl-2 expression significantly, in the well accepted view of that (-)gossypol interrupted function of Bcl-XL and Bcl-2 by interacting with them, we confer that in K562 cells (-)gossypol also lower the function of Bcl-2 as well as Bcl-XL which is overexpressed in Bcr-Abl positive cells. Combination with (-)gossypol and imatinib leaded to more extensive changes in Bcr-Abl signaling and apoptosis signaling in K562 cells, including downregulation of Bcl-Xl, Mcl-1, Bcl-2, Bcr-Abl, JNK by a varied extent. Combination treatment seemed to have less effect on cell cycle, although (-)gossypol caused G0/G1 arrest, which may partly owe to the induction of P21.2. The establishment of K562/R cell line and inhibition of (-)gossypol in K562/R cells.By increasing imatinib concentration in K562 cell culture step by step, an imatinib resistant K562 cell line was established, referred as K562/R. In contrast with K562 cell line, the sensitivity of K562/R to imatinib decreased 15-fold, while both cell lines had similar responses to (-)gossypol. By comparison, expression of Bcr-Abl and mitochondrial Bcl-2 dramatically increased, Mcl-1 was unchanged. However, Bcl-XL decreased to an almost undetectable extent perplexingly. Anyway, because of the complex mechanisms in imatinib resistance further identifications between the two cell lines need to be done.In vivo result from nude mice bearing hollow fibers showed that (-)gossypol inhibited growth of K562/R cells in nude mice. Compared with control group the dose of 15mg/kg and 30mg/kg had P values less than 0.05. Morphologically, (-)gossypol induced typical apoptotic change -- karyorrhexis in K562/R cells at concentration of 10μmol/L. Flow cytometric assay was performed to evaluate the cell cycle and apoptosis. Results showed that being treated with (-)gossypol, percentage of apoptotic K562/R cells increased, and G0/G1 arrest of cell cycle in K562/R cells was observed with a dose-dependent manner. Loss ofΔΨm was also detected in (-)gossypol treated K562/R cells, (-)gossypol caused cytochrome C release was proved by using western blot. To investigate the mechanism of growth inhibition and apoptosis of (-)gossypol in K562/R cells, protein and mRNA expression of several Bcl-2 family members were analyzed using western blot and RT-PCR assays, (-)gossypol decreased Bcl-XL and Mcl-1 protein level, and increased mRNA level of Bax, Bad and Bak. Unlike in K562 cells (-)gossypol seemed to have no effect on P21 induction in K562/R cells(data not shown).Conclusively, (-)gossypol inhibited K562/R by inducing apoptosis and G0/G1 arrest of cell cycle, (-)gossypol induced apoptosis by decreasing antiapoptosis proteins and increasing proapoptosis gene transcription. (-)gossypol caused G0/G1 arrest may have no relationship with P21.In summary, (-)gossypol may be effective in CML treatment and combining it with imatinib may be more promising, although there is still more to be explored before (-)gossypol can be used in clinic. Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative effects on many kinds of cancer cells in vitro. Here, we tested the antiproliferative effect of gossypol and its derivative gossypolone. The results showed that the rank of intensity was (-)gossypol, gossypolone, (±)gossypol, (+)gossypol. (-)gossypol induced apoptosis in human melanoma cell line A375, but did not cause cell cycle change. (-)gossypol increased P53 protein level in A375, which may contribute partly to the apoptosis, but it had no obvious effect on Bcl-2 mRNA expression. We also found that (-)gossypol lowered antimetastatic ability of B16BL6 cells.
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