Taxol induced apoptosis regulates amino acid transport in breast cancer cells.
A major outcome from Taxol treatment is induction of tumor cell apoptosis. However, metabolic responses to Taxol-induced apoptosis are poorly understood. In this study, we hypothesize that alterations in specific amino acid transporters may affect the Taxol-induced apoptosis in breast cancer cells. In this case, the activity of the given transporter may serve as a biomarker that could provide a biological assessment of response to drug treatment. We have examined the mechanisms responsible for Taxol-induced neutral amino acid uptake by breast cancer cells, such as MCF-7, BT474, MDAMB231 and T47D. The biochemical and molecular studies include: (1) growth-inhibition (MTT); (2) transport kinetics: (3) substrate-specific inhibition; (4) effect of thiol-modifying agents NEM and NPM; (5) gene expression of amino acid transporters; and (6) apoptotic assays. Our data show that Taxol treatment of MCF-7 cells induced a transient increase in Na(+)-dependent transport of the neutral amino acid transporter B0 at both gene and protein level. This increase was attenuated by blocking the transporter in the presence of high concentrations of the substrate amino acid. Other neutral amino acid transporters such as ATA2 (System A) and ASC were not altered. Amino acid starvation resulted in the expected up-regulation of System A (ATA2) gene, but not for B0 and ASC. B0 was significantly down regulated. Taxol treatment had no significant effect on the uptake of arginine and glutamate as measured by System y(+) and X(-) (GC) respectively. Tunel assays and FACS cell cycle analysis demonstrated that both Taxol- and doxorubicin-induced upregulation of B0 transporter gene with accompanying increase in cell apoptosis, could be reversed partially by blocking the B0 transporter with high concentration of alanine, and/or by inhibiting the caspase pathway. Both Taxol and doxorubicin treatment caused a significant decrease in S-phase of the cell cycle. However, Taxol-induced an increase primarily in the G2 fraction while doxorubicin caused increase in G1/G0 together with a small increase in G2. In summary, our study showed that Taxol induced apoptosis in several breast cancer cells results in activation of amino acid transporter System B0 at both gene and protein level. Similar response was observed with another chemotherapeutic agent Doxorubicin, suggesting that this increase is in response to apoptosis, and not only due to changes in cell cycle related events.
Wu
Yanyuan
Y
Division of Cancer Research and Training, Department of Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, CA 90059, USA.
Shen
Dejun
D
Chen
Zujian
Z
Clayton
Sheila
S
Vadgama
Jaydutt V
JV
D000409:Alanine / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D000596:Amino Acids / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001943:Breast Neoplasms / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D020287:In Situ Nick-End Labeling; D008301:Maleimides / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism; D013217:Starvation
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D008301:Maleimides; D004317:Doxorubicin; D012964:Sodium; C028525:N-phenylmaleimide; D020169:Caspases; D005033:Ethylmaleimide; D000409:Alanine; D017239:Paclitaxel