Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: Crosstalk between ODC and BCL-2

Abstract

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Therapeutic mechanisms of granulocyte-colony stimulating factor in methotrexate-induced parotid lesion in adult rats and possible role of telocytes: A histological study

“…It was evidenced to reduce intracellular glutathione-based antioxidants and produce oxygen free radicals during its intracellular metabolism. This oxidant/antioxidant imbalance leads to lipid peroxidation followed by organelles and plasma membranes lysis [39,40] . Such lipid peroxidation was enforced by significantly increased MDA value, acinar and ductal cell membrane rupture and ductal epithelial discontinuity in MTX group.…”

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases’ cascade

“…In our previous studies, overexpression of ODC represses the extrinsic death pathway of TNFα-induced apoptosis [4] and blocks the intrinsic pathway of chemotherapeutic drug-induced apoptosis [5]. In addition, overexpressed ODC up-regulates the expression of Bcl-2 to enhance the anti-apoptotic effect of ODC [6]. Therefore, we are interested in the molecular and cellular function of the natural ODC inhibitor, antizyme, in the apoptosis of haematopoietic cells.…”

“…The supernatant was boiled in loading buffer and an aliquot corresponding to 100 µg of protein was separated by SDS-PAGE. After blotting, the membranes were incubated with anti-antizyme (MDBio, TWN), anti-ODC, anti-p53, anti-p21, anti-Bax, anti-Bcl-2, anti-Bcl-xL, anti-cyclin E, anti-cyclin D, anti-cyclin A, anticyclin B, anti-cyclin-dependent kinase 1 (Cdk1), anti-Cdk2, anti-Cdk4, anti-cytochrome c, anti-caspase 9, anti-caspase 3, anti-Apaf-1, anti-PARP and anti-actin antibodies (Lab Vision and Santa Cruz, CA) for 6 h and the second antibody labeled with horseradish-peroxidase was adjacently incubated Enzyme activity assay of ODC ODC enzyme activity was assayed at 37 • C by measuring its product, putrescine, as described previously [6]. Samples were suspended in ODC buffer ( …”

“…ODC is a pyridoxal 5 -phosphate dependent enzyme that catalyzes the decarboxylation of ornithine to putrescine, a rate-limiting step in the formation of polyamines (putrescine, spermidine and spermine) [2]. ODC and polyamines play a critical role in normal biological functions, including embryonic development, cell cycle, proliferation, differentiation and apoptosis [3][4][5][6]. They are associated with the human diseases including cancers (skin, breast, lung, upper respiratory tract, oesophagus, liver, forestomach, large bowel, colon, rectum, prostate, neuron, brain, urinary bladder, melanoma and myeloma, etc), diabetes, rheumatoid arthritis, systemic lupus erythematosus, early diabetic renal hypertrophy, cardiac hypertrophy, cerebral ischemia and Alzheimer's disease [7][8][9][10][11][12][13][14][15].…”

Novel roles of prolactin and estrogens in breast cancer: resistance to chemotherapy

“…Another group reported that pretreatment of ovarian carcinoma cells with PRL inhibits cisplatin-induced cell death (Asai-Sato et al 2005). PRL also antagonizes apoptosis caused by methotrexate, an antifolate agent, in human promyelocytic leukemia HL-60 cells (Hsu et al 2006).…”