| BackgroundLeukemia is malignant tumor of hematopoietic system. The major treatments of leukemia are chemotherapy and bone marrow transplantation. Because of drug resistance to some chemotherapy drugs and the side effects of chemotherapy, leukemia has poor prognosis. Thus, it is urgent for us to find another effective therapy.Diallyl disulfide (DADS) is a fat-soluble, effective component of garlic. It has been shown that DADS could decrease the formation of carcinogen-induced cancers and to inhibit the proliferation of various types of cancer cells, such as prostate cancer, liver cancer, lung cancer, gastric cancer and colon cancer. The main mechanisms of DADS effect on cancer include that suppression of the formation of DNA adducts, antioxidant formation, regulation of cell-cycle arrest, induction of apoptosis and cell differentiation, histone modification, and inhibition of angiogenesis and cell invasion.Tumor cells, unlike the normal differentiated counterparts, have elevated rates of glucose uptake and lactic acid production in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, allows tumor cells to function like fetal cells and to use a large fraction of glucose metabolites to synthesize macromolecules (such as amino acids, phospholipids, and nucleic acids), which support tumor cell growth. Pyruvate kinase regulates the final rate-limiting step of glycolysis, which catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of adenosine triphosphate (ATP). Christofk found that the increased expression of PKM2 may lead to the abnormal metabolism of aerobic glycolysis of tumor cells. It was first demonstrated that the knockdown of PKM2 in a panel of cancer cell lines decreased the rate of glycolysis and proliferation by Christofk and colleagues in 2008. Introducing PKM2 but not PKM1 to the knockdown cells not only enhanced glycolysis but also increased the ability to form xenograft tumors. PKM2 is highly expressed in cancer cells. PKM2 have two forms:dimers and four polymers. Gao reported that PKM2 could act as a protein kinase. PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. The nuclear PKM2 is a dimeric form. Yang reported that the activation of epidermal growth factor receptor (EGFR) induced nuclear translocation of PKM2 and induced the tumor growth.Thus, whether the effect of DADS on the proliferation of leukemia cells? Is the tumor cell proliferation inhibition related to PKM2? Can the DADS influence the expression and nuclear entry of PKM2? Has yet to see the relevant reports.MethodsPart IWestern blot was used to detect the expression levels of PKM2 protein in different leukemia cell lines, and to find out the leukemia cell line which was highly expressed PKM2; The growth inhibitory effect of DADS on the cell line was measured by cell counting kit-8 (CCK-8) assay. The effect of DADS on cell cycle and apoptotic were detected by flow cytometry (FCM). Hochest33258 staining was used to examine DADS-induced apoptosis in human leukemia cell line. RT-PCR was used to detect mRNA expression of CCND1 and c-Myc. Western blot was used to detect the expression of Bax> Bcl-2、Cleaved-caspase 3、CyclinD1、P21 proteins. In addition, western blot was used to detect the effect of DADS on the protein expression levels of PKM2 in cytoplasm and nucleus. In addition, the distribution of PKM2 in cells after treated with DADS was detected by immunofiuoresence staining.Part IITo further clarify the mechanism of the effect of DADS on PKM2, western blot was used to detect the effect of DADS on the expression of P-EGFR, P-ERK, P-MEK1/2, EGFR, ERK, MEK, PIN1, (3-catenin proteins. And detect the effects on PKM2 nuclear import pathway treated with AG-1478 (6.25μM) and DADS (100μM) alone or combination.Results1. KG1-a cell was the leukemia cell line which highly expressed PKM22. DADS had the most efficient inhibitory effect on KG1-acells.3. Cell cycle:DADS could arrest the cell cycle in GO/G1 phase significantly.The percentages of cell cycle in GO/G1 phase in KG1-a cells increase by DADS were (41.19±0.05)%, (54.32±0.02)% and (68.70±0.02)% respectively. (P<0.05)4. Cell apoptosis:DADS have inhibitory effect on KG1-α cell growth. The percentages of early apoptosis in KG1-α cells induced by DADS were (1.98±0.23)%, (17.75±0.80)% and (23.01±1.27)% respectively. The proportions of early apoptotic cells treated by DADS increased in a dose-dependent manner.5. Hoechst 33258 staining indicated that the typical apoptosis morphological changes could be found in KG1-α cells induced by DADS.6. The results of RT-PCR showed that the expression of CCND1, c-Myc mRNA were dropped significantly.7. The results of Western blotting showed that DADS could reduce the accumulation of PKM2 in nucleus; it had no obvious effect on the expression of PKM2 in the cytoplasm. The expression of anti-apoptotic protein Bcl-2 was dropped significantly. In addition to, the protein levels of Bax, Cleaved Caspase-3, P21, CyclinD1 were up-regulated by DADS.8. The results of Western blotting showed that the expression of phosphorylated of EGFR, ERK MEK1/2 and PIN1 were dropped significantly by DADS, but it had no obvious effects on the expression of EGFR, ERK1 and MEK. The expression level of PKM2 significantly decreased in the nucleus by AG-1478 in combination with DADS. When cells treated with or without AG-1478 in combination with DADS, it was indicated that DADS have effect on the levels of phosphorylated of EGFR, ERK, MEK 1/2 and β-catenin more significantly.Conclusion1. KG1-α cell was the leukemia cell line who highly expressed PKM2. DADS had the most efficient inhibitory effect on KG1-a cell.2. DADS could arrest the cell cycle in G0/G1 phase, inhibit the proliferation, and and promote apoptosis through the up-regulation of Bax, P21, Cleaved-Caspase 3 proteins and down-regulation of Bcl-2, CyclinDl proteins.3. DADS can effect on the EGFR/ERK/PKM2 signaling pathway, abrogated EGF-induced nuclear accumulation of PKM2. |
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