Regulation of the multidrug resistance transporter P‐glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species

Abstract

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50 -100 m) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42°C, whereas only moderate elevation of P-glycoprotein was found in large (350 -450 m) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia-inducible factor 1␣ (HIF-1␣) was observed. Elevation of HIF-1␣ and Pglycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1␣ and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1␣ and P-glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1␣ is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1␣ under control conditions. Key words: multidrug resistance; P-glycoprotein; hyperthermia; hypoxia-inducible factor 1-␣ Hyperthermia in combination with chemotherapy and radiotherapy has been previously successfully used in anti-cancer strategies. [1][2][3][4][5] The efficiency of chemotherapeutic as well as radiotherapeutic cancer treatment is generally restricted by the expression of MDR transporters that confer resistance to a variety of structurally unrelated, clinically important antineoplastic agents. 6 -8 The most clinically significant MDR transporter P-glycoprotein, a 170 kDa ATP-dependent membrane-bound transporter, has been recently demonstrated to be upregulated by hyperthermia. 9,10 The promotor of the mdr-1 gene, which encodes for P-glycoprotein, harbors different responsive elements that are inducible by various stress factors including hyperthermia. 11 In this respect, it has been previously demonstrated in colon cancer cells that hyperthermia resulted in nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of mdr-1. 12 Binding of hyperthermia factors to defined heat shock element (HSE) motifs of the mdr-1 pr...

NADPH oxidase-mediated reactive oxygen species production activates hypoxia-inducible factor-1 (HIF-1) via the ERK pathway after hyperthermia treatment

“…Meanwhile, HT-regulated induction of HIF-1 activity could be either beneficial or detrimental. Although we suggest that HTinduced HIF-1 up-regulation may play a role in regulating tumor reoxygenation, its possible involvement in drug resistance by upregulating p-glycoprotein was also reported by Wartenberg et al (27). Thus, to preferentially take advantage of HT-induced HIF-1 activation and also suppress its deleterious effects, further studies are needed to investigate how HIF-1 activation is regulated by combined treatments of HT, radiation, and chemotherapy.…”

“…Although 1 h of heat treatment down-regulates HIF-1α and VEGF levels in murine macrophages under hypoxia (25), in tumor cells heat increases HIF-1α independently of oxygen (26,27). However, the downstream effects of HT-induced HIF-1α up-regulation have not been fully validated.…”

Hypoxia-induced increases in A549/CDDP cell drug resistance are reversed by RNA interference of HIF-1α expression

“…Furthermore, increases in HIF-1α expression were positively correlated with those of MDR1 and MRP. Consistent with our current findings, Wartenberg et al found that HIF-1α expression was increased in cancer cells under hypoxic conditions, leading to the up-regulation of MDR1 (10). Furthermore, in A549 cells, a positive correlation between HIF-1α and MDR1, and MRP expression has also been observed (4), suggesting that hypoxiainduced drug resistance in A549/CDDP cells may be due to the HIF-1α-mediated upregulation of MDR1 and MRP.…”

PolyHEMA spheroids are an inadequate model for the drug resistance of the intractable solid tumors

“…As shown in Figure 1B, HCT 116 cells grown in monolayer have no detectable E-cadherin, whereas both day 3 and day 6 spheroids show robust protein signals with the day 6 signal being somewhat the stronger, confirming the previous findings of Mueller et al 30 Many studies have shown that spheroid grown cells have high levels of multidrug resistance transporters. [33][34][35] To exclude the possibility that expression of these proteins was the basis of the drug resistance found in the spheroid model (particularly since we were using a colon carcinoma cell line) we performed immunoblot analysis for several multidrug resistance transporters. As shown in Figure 1B, expression of the three transporters P-gp, MRP-1 and ABCG2…”