An association of Bcl‐2 phosphorylation and Bax localization with their functions after hyperthermia and paclitaxel treatment

Abstract

Apoptosis is induced by many kinds of therapy-related inducers, such as hyperthermia and chemotherapeutic agents. However, differences in apoptotic pathways between these inducers remain unclear, although knowing the differences is important to map out a therapeutic strategy. Therefore, we focused on the localization and phosphorylation of Bcl-2 and Bax, key mediators of the apoptotic pathway, after hyperthermia and paclitaxel treatment of PC-10 squamous cell carcinoma cells that excessively expressed Bcl-2 and Bax in the cytoplasm. Paclitaxel treatment markedly induced qualitative changes in Bcl-2, whereas hyperthermia did only quantitative changes in Bax. The levels of Bax increased gradually with the duration of hyperthermia, whereas Bcl-2 levels slightly decreased. On the other hand, paclitaxel treatment induced dose-and time-dependent phosphorylation of Bcl-2. Interestingly, phosphorylated Bcl-2 was observed in the specific subcellular sites, mitochondria-and lysosomerich fractions. Both treatments disturbed the heterodimerization of Bax with Bcl-2. Hyperthermia, but not paclitaxel treatment, induced a gradual Bax translocation from the cytoplasm to the nucleus. Although both treatments induced a prominent cell cycle disturbance in the G2M phase, paclitaxel treatment induced typical apoptosis, and hyperthermia hardly induced apoptosis. Our results suggest that the subcellular redistribution of Bax and the phosphorylation of Bcl-2 depend on the type of apoptosis inducers, such as hyperthermia and paclitaxel, and Bcl-2 has a central role in the decision of apoptotic outcome. Our data may afford new insights in apoptosis from the aspect of an association of Bcl-2 phosphorylation with intracellular Bax localization. © 2002 Wiley-Liss, Inc. Key words: Bcl-2; Bax; hyperthermia; paclitaxel; lung cancer cellsPaclitaxel, microtubule-stabilizing agents, demonstrates a marked activity against several kinds of tumors 1 by arresting cell-cycle progression and inducing apoptosis. 2-4 Mild hyperthermia disturbs functions of the microtubule system and triggers apoptosis as well. 5 In addition, local or systemical hyperthermia is used in conjunction with paclitaxel in cancer tretment, 6 but whether a combined thermo-chemotherapy enhanced 5,7,8 or inhibited 9,10 the susceptibility of cells to apoptotic stimuli is still controversial. Therefore, understanding how paclitaxel and hyperthermia modalities induce apoptosis is critical to map out a better anticancer strategy that may offer synergistic benefits when used concurrently.Cellular apoptotic potential is mainly regulated by the relative levels of Bcl-2 and Bax, Bcl-2 family members with opposite functions and their interactions. 11 These proteins mainly act on mitochondria at a critical decision point to irreversible cellular damage. 12-15 The antiapoptotic Bcl-2 suppresses apoptosis, whereas the proapoptotic Bax counteracts the Bcl-2 function to accelerate apoptosis. These proteins exert their activity via protein/ protein interactions, such as homodimerization and heter...

Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5

“…There is a large literature on this topic, but no clear and general answers have emerged. We were also unable to obtain a clear-cut answer thus far, and our data failed to support some previous suggestions (19)(20)(21). We did confirm that two stressactivated protein kinase pathways, JNK and p38, both previously implicated in paclitaxel-induced cell death (22)(23)(24), were activated by antimitotics, and to some extent, the degree of activation correlated with drug sensitivity.…”

“…However, a few studies reported death by poorly understood caspase-independent mechanisms (17,18). Inactivation of antiapoptotic proteins Bcl2 and Bcl-xL by phosphorylation was implicated in death triggered by paclitaxel (19,20), as were several kinases, including c-Jun NH 2 terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen activated protein (MAP) kinase, AKT, and protein kinase A (21-26). There are contradictory reports regarding the role of the spindle assembly checkpoint in mediating cell death triggered by antimitotics.…”

Magnetic thermoablation stimuli alter BCL2 and FGF-R1 but not HSP70 expression profiles in BT474 breast tumors

“…18 In vitro BCL2 protein is known to be slightly downregulated in tumor cells immediately after heat stress (180 minutes, 42.5°C). 15,16 Contrarily, the treatment of human colorectal tumor (RKO.C) and isogenic RC10.1 cells to lower temperatures such as 1 hour at 42°C (water bath hyperthermia) did not reveal any changes in BCL2 expression. 17 Presumably, faster and more direct heat generation in tumor tissues (magnetic heating vs water bath hyperthermia) as well as a distinct threshold of temperature dosage is required for a reduction of BCL2 expression, which is readily provided by the utilization of thermoablative stimuli.…”

“…[15][16][17][18] In general, the expression of BCL2, FGF-R1, and HSP70 has been mainly elucidated in connection with hyperthermic temperatures up to 43°C only.…”

Taxol Induces Caspase-10-dependent Apoptosis

“…Therefore, cells treated with taxol are unable to proceed normally through the cell cycle and are arrested in the G 1 and G 2 /M phases (3)(4)(5). During the last decade, additional activities of taxol have been described including 1) its effect on cell signaling and gene expression, activation of mitogen-activated protein kinases, Raf-1, proteintyrosine kinases, and c-Jun NH 2 -terminal kinase (6 -11), and 2) its effect on triggering apoptosis by caspase-dependent and independent pathways (12)(13)(14)(15) as well as necrosis (16) and regulating the expression of apoptosis-related proteins like Bcl-2, Bad, Bcl-xL, p21 WAF-1/CIP-1 , tumor necrosis factor-␣ (TNF-␣) receptor 1 (TNFR1), and the tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 (17)(18)(19)(20)(21)(22). Up-regulation of the transcription factor FoxO3a by taxol in MCF-7 breast cancer cells can also result in increased levels of Bim, a proapoptotic BH3-only Bcl-2 family member, which can cause apoptosis in these cells (23).…”