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Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line
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Toxicology in Vitro 22 (2008) 873–878 www.elsevier.com/locate/toxinvit
Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line Fa´bio Klamt a,*, Daniel Thompsen Passos b, Mauro Antoˆnio Alves Castro a,b, Daniel Pens Gelain a, Ivana Grivicich b, Jose´ Cla´udio Fonseca Moreira a a
Centro de Estudos em Estresse Oxidativo, Departamento de Bioquı´mica, ICBS/Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ramiro Barcelos 2600 – anexo, Porto Alegre, RS 90035-003, Brazil b Universidade Luterana do Brasil (ULBRA), Canoas, Brazil Received 7 November 2007; accepted 7 January 2008 Available online 15 January 2008
Abstract Multidrug resistance (MDR) is the major obstacle to cancer chemotherapy. MDR phenotype is mainly related to the over-expression of MDR1 gene, being responsible for tumor resistance to several chemotherapeutic drugs. It has been suggested that MDR1 expression is redox-regulated and we have recently described a pro-oxidative effect of retinol. Here we tested the therapeutic use of retinol as a modulator of MDR1 gene expression in tumor cell lines, and verified in situ the enhancement of anticancer drug efficacy. Two human colorectal adenocarcinoma cell lines (HT29, SW620) with different degrees of MDR1 expression were used. Cells were pre-treated with a sublethal dose of retinol and then challenged with the etoposide (VP16) drug. The drug GI50 was assessed by SRB method and levels of MDR1 expression were determined by semi-quantitative rtPCR. Retinol treatment caused a 40% decrease in MDR1 expression and increased VP16 toxicity. MDR1 expression and drug sensitivity were restored to control values when mannitol (a hydroxyl radical scavenger) was co-administrated. Our data point a role to the use of retinol as an adjuvant in the treatment of tumors with MDR phenotype. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Multidrug resistance; Retinol; MDR1; Chemotherapy; Oxidative stress
1. Introduction The resistance of tumor cells to chemotherapeutic interventions remains a major cause of treatment failure in cancer patients. During the past two decades, the research on resistance to cancer chemotherapy was mainly focused on multidrug resistance (MDR). In humans, this phenotype of tumor cells is clinically associated with the over-expression of MDR1 gene and enhancement of drug efflux mediated by P-glycoprotein (P-gp) (Varma et al., 2003). P-gp is a member of the ATP-binding cassette superfamily of transporter proteins, which can export a wide range of chemotherapeutic agents not structurally-related (e.g., vim-
*
Corresponding author. Tel.: +55 51 3308 5577; fax: +55 51 3308 5535. E-mail address: [email protected] (F. Klamt).
0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.01.004
blastine; incristine; doxorubicin; daunomycin; etoposide and others). This action of P-gp contributes to the tumor’s intrinsic or acquired overall resistance against chemotherapy. P-gp expression is positively correlated with drugresistance in cancers of the colon, kidney and adrenal gland (Tsuruo et al., 2003). Besides the great effort to inhibit MDR1 expression in resistant tumor cells, no successful therapeutic proceeding has been developed so far. Moreover, MDR treatment is based on the use of classical inhibitors of P-gp activity (e.g., cyclosporine A; verapamil) but the high incidence of undesired side-effects of these drugs tends to compromise their clinical use (Krishna and Mayer, 2001; Teodori et al., 2002). A more efficient strategy to circumvent the MDR phenotype would be the down-regulation of gene expression coding for the transporters. Recently, it has been pointed out that the expression of P-gp may be
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redox-regulated, which led to new perspectives to the search for new mechanisms to downplay MDR phenotype in cancer cells (Wartenberg et al., 2001). Retinoid supplementation has been widely used therapeutically in the prevention and treatment of several types of diseases, particularly epithelial cancers and leukemia, including both pre-malignant and malignant tumors (Blomhoff, 1994). Retinoid action is classically mediated by specific nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) belonging to the steroid/thyroid superfamily of transcription factors (Altucci and Gronemeyer, 2001). In addition to these classical nuclear-dependent mechanisms, recent works suggested that retinoids may act directly on the modulation of different redox-regulated enzymes, such as Protein Kinase C (PKC) isoforms, serine/threonine kinases, ornithine decarboxilase (ODC) and constitutive Raf (cRaf) (Hoyos et al., 2000; Klamt et al., 2000). Moreover, a growing body of evidence has demonstrated that b-carotenoids, vitamin A and retinoids have pro-oxidant properties (Klamt et al., 2003; Dal-Pizzol et al., 2000a,b; Murata and Kawanishi, 2000). Retinol causes an increase in 8-oxo,dGuanosine adducts in the DNA of treated cells, suggesting the generation of high reactive hydroxyl radical (OH) by retinol treatment (Dal-Pizzol et al., 2000a). As demonstrated, once formed, free radicals can act as cellular second messengers, regulating several signal transduction pathways and consequently, gene expression (Frota et al., 2004). The experiments presented in this report were designed to investigate both (1) if retinol supplementation is able to modulate MDR1 gene expression and (2) if modulation of MDR1 causes an increase in anticancer drug efficacy. Our results provide evidence for the involvement of free radical generation as a possible mechanism of retinol action, as co-treatment with mannitol restored MDR1 expression and drug sensitivity to control values. The data are discussed with reference to the use of retinol supplementation as an adjuvant during cancer chemotherapeutic interventions.
by sulforhodamine B (SRB) method, as described in ‘‘Cell Growth Inhibition Measurements”, and MDR expression was assessed by semi-quantitative rtPCR, as described below. The dose and time exposure of retinol chosen (7 lM, 24 h) were based on the following previous observations: (i) this concentration increases free radicals production and activates redox signaling in different experimental models (Klamt et al., 2000; Dal-Pizzol et al. 2000b; Klamt et al., 2003); (ii) it was the concentration observed to abolish MDR1 and MDR3 gene expression in cultured rat Sertoli cells (Frota et al., 2004); and (iii) data depicted at Fig. 1 (results section) showed that these conditions did not induce cytotoxicity in SW620 and HT29 cell lines. 2.2. Cell growth inhibition measurements
2. Materials and methods
Cells were separated into a single-cell suspension in culture medium by trypsinization and seeded into 96-well culture plates at a density of 7.5 103 cells/cm2 (initial cell density). Cell growth was performed at 37 °C in a 5% CO2 humidified atmosphere. The medium was replenished each 48 h after seeding and the cell number was determined periodically by the sulforhodamine B (SRB) staining assay. Briefly, adherent cell cultures were fixed in situ by adding 50 ll of cold 50% (w/v) trichloroacetic acid (TCA) (final concentration, 10% TCA) for 60 min at 4 °C. The supernatant was then discarded, and the plates were washed five times with deionized water and dried. One hundred microlitres of SRB solution (0.4% in 1% acetic acid, w/v) was added to each microtitre well and the culture was incubated for 10 min at room temperature (25 °C). Unbound SRB was removed by washing five times with 1% acetic acid and the plates were air dried. The bond stain was solubilized with Tris buffer, and the absorbance was read on an automated spectrophotometric plate reader at a single wavelength of 515 nm. Cell growth was assessed in the SRB-staining assay by whole culture protein determination, showing sensitivity and reproducibility. The GI50 (growth inhibition index) value was obtained by determining the concentration of retinol that inhibited cell growth by 50% using this method.
2.1. Cell cultures and treatments
2.3. RNA extraction and PCR
The human colon adenocarcinoma cell lines SW620 and HT29, which have different degrees of MDR1 ABCB1; ATP-binding cassette, sub-family B member (1) gene expression were obtained from the American Type Culture Collection (ATCC). Basal levels of MDR1 gene expression were obtained based on the analysis of the cDNA microarray gene expression Serial Analysis of Gene Expression (SAGE) databank from the NCI’s Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/SAGE) (Fig. 1A). Cells in the exponential growth phase were routinely cultured, pre-treated with retinol 7 lM by 24 h and then exposed to different concentrations of etoposide (VP16) for additional 72 h. Cell growth inhibition was assessed
Total cellular RNA was isolated by the guanidinium isothiocyanate/phenol–chloroform method (Chomczynski and Sacchi, 1987). In each experiment, water was used as a negative control for contamination. Reverse transcription of 1 lg of the total RNA using 10 pmol of specific 30 primer was performed for 50 min at 42 °C using 20 lL of reverse transcriptase buffer (Gibco/BRL) containing 1 mM each of dATP, dGTP, dCTP, and dTTP, and 200 U of AMV reverse transcriptase (Gibco–BRL), as described (Murphy et al., 1990). The samples were then heated at 95 °C for 5 min to terminate the reverse transcription. The resulting cDNA was serially diluted in water from 1:2 (500 ng of RNA) to 1:32 (31.25 ng of RNA). To these dilutions were
F. Klamt et al. / Toxicology in Vitro 22 (2008) 873–878
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Fig. 1. (A) Basal MDR expression levels were examined in HT29 and SW620 cells by meta-analysis of cDNA microarray gene expression SAGE databank from the NCI’s Cancer Genome Anatomy Project. (B) GI50 values of retinol on both cell lines were calculated as described in Section 2. SW620 cells were treated with retinol 7 lM or retinol plus mannitol 1 mM for 24 h and MDR1 expression levels were evaluated by semi-quantitative rtPCR (C and D). Results are expressed as mean ± SEM. * Significantly different from respective control (p < .05).
added 50 lL of PCR buffer (Promega) containing 200 lM dNTP, 2.5 lL of the cDNA template, 1.25 U of Taq DNA polymerase (Perkin–Elmer Cetus), 1 lM of MDR1 primers, and 0.5 lM of b-actin primers. Primers were selected from two exons separated by one or more long intronic sequences, which allowed identification of contaminating genomic DNA. The sequences of primers used were as following: MDR1 (PCR product = 157 bp) 50 -CCCATCATTgCAATAgCAgg-30 – forward and 50 -gTTCAAACTTCTgCTCCTgA-30 – reverse; b-actin (PCR product = 228) 50 -CgggAAATCgTgCgTgACAT-30 – forward and 50 -ggAgTTgAAggTAgTTTCgTg-30 – reverse). The reaction mixture was overlaid with 100 lL of mineral oil, and heated at 95 °C for 2 min and carried out for 30 cycles. One cycle consisted of denaturation at 94 °C for 1 min, 15 s; annealing at 55 °C for 1 min, 15 s; and extension at 72 °C for 2 min. The PCR products were subjected to electrophoresis in 1X Tris-borate/2 mM EDTA buffer though 2% agarose containing ethidium bromide for UV visualization. 2.4. Data analysis PCR products intensities were analyzed by scanning densitometry (ImageJ 1.36b; NIH, USA), using b-actin as housekeeper gene control. The results were plotted as densitometric arbitrary units against the quantity of RNA present in the PCR reaction diluted from the reverse transcriptase reaction. For GI50 drug values, the results were
expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA), using a Neuman–Keuls test to compare means values across groups. Differences were considered to be significant when p < .05. 3. Results Meta-analysis of cDNA microarray gene expression revealed that MDR1 expression values in SW620 cells are significantly increased compared to HT29 cells (Fig 1A). This data indicated that these two cell lines are suitable to compare the effects of chemotherapeutic drugs on different human colon adenocarcinoma cell lines, one presenting low MDR1 expression (HT29) and the other with high expression levels of MDR1 (SW620). Next, we evaluated the GI50 values for HT29 and SW620 cells treated with varying concentrations of retinol (up to 40 lM). With concentrations up to 10 lM, retinol caused no statistically detectable growth inhibition in both cell lines (Fig. 1B). However, retinol 20 lM induced a significant decrease (40%) in SW620 cell number, with a further decrease observed at 40 lM. The number of HT29 cells was only decreased by retinol at 40 lM (Fig. 1B). These results indicated that concentrations up to 10 lM of retinol do not affect cell viability. Next, we evaluated the effect of retinol on MDR1 gene expression in both cell lines. As expected, semi-quantitative rtPCR analysis did not found detectable MDR1 expression levels in HT29 control cells, and retinol did not change this
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Fig. 2. HT29 (A) and SW620 (B) cells pre-treated with retinol 7 lM or retinol plus mannitol 1 mM were subjected to VP16 treatment and cell viability was evaluated. Results are expressed as percentage of control (mean ± SEM). * Significantly different from respective control (p < .05).
profile (not shown). In the MDR1-expressing SW620 cells, however, retinol incubation significantly decreased MDR1 expression to levels approximately 50% of control cells (Fig. 1C and D). To investigate if MDR1 inhibition by retinol was mediated by a redox-dependent mechanism, we evaluated the effect of the antioxidant mannitol, a classical hydroxyl radical scavenger, on the effect of retinol upon MDR1 expression in SW620 cells. Mannitol co-administration restored MDR1 gene expression to control values (Fig. 1C and D); ethanol (the retinol vehicle), or mannitol alone, had no effect on the degree of MDR1 gene expression (data not shown). These results indicated that MDR1 down-regulation by retinol in SW620 cells is mediated by a redox-dependent mechanism. Next, to assess if the retinol-mediated decrease in MDR1 gene expression cause an increase in the cellular retention of anticancer drug, we determined the GI50 values of the anticancer compound etoposide (VP16, which is a classical P-gp substrate) in HT29 and SW620 cells pre-treated with retinol. VP16 concentration curves (0–20 lM) showed no differences on inhibition of cell growth between untreated and retinol pre-treated HT29 cells, and addition of the antioxidant mannitol to the pre-treatment also presented no effect (Fig. 2A). In contrast, retinol pre-treated SW620 cells were more susceptible to the inhibition effect of increasing concentrations of VP16 (Fig. 2B); the antioxidant co-treatment with mannitol reversed the effect of retinol, indicating the involvement of a redox-dependent mechanism on this effect. Calculated GI50 values of VP16 on both cell lines pre-treated with retinol are depicted at Table 1. Since these cells express high levels of MDR1, and this expression is inhibited by retinol in a redox-dependent way (Fig. 1), this result indicates that retinol pre-treatment increases SW620 susceptibility to VP16 through an oxidant down-regulation of MDR1. 4. Discussion Retinoid-based therapies present potential perspectives for the treatment of different types of diseases related to cell
Table 1 GI50 (lM) values for etoposide (VP16) in human neoplasic cell lines exposed for 72 h, pre-treated with 7 lM of retinol Treatments
Cell lines HT29
SW620
VP16 VP16 pre-treated with retinol VP16 pre-treated with retinol + mannitol
1.15 ± 0.23 0.93 ± 0.14 1.08 ± 0.11
0.64 ± 0.12 0.20 ± 0.07a 0.57 ± 0.18
Two human adenocarcinoma cell lines (ATCC) were selected by different degree in MDR1 expression, and cultivated as described in ‘‘Materials and Methods” section. HT29 (with undetected MDR1 gene expression) and SW620 human adenocarcinoma cell line (with higher level of MDR1 expression) were pre-treated with 7 lM of retinol for 24 h, washed, exposed to different concentrations of VP16, and the GI50 of the drug was determined. Data represent means ± SD of three independent experiments carried out in triplicates. a Different from VP16-treated cells and from VP16 pre-treated with retinol + mannitol cells (p < .01).
cycle disruption/cell death and increased ROS formation. Diseases such as skin cancer, lung cancer, Parkinson’s disease and Alzheimer disease, among others, were suggested to be treatable by retinoid-based therapies (Njar et al., 2006; Greenwald et al., 2006; Okuno et al., 2004; Ono and Yamada, 2007). Protocols for treatment of malignant diseases such as leukemia and lung cancer have been proposed and clinical trials have been carried out (Fenaux et al., 2007; Kakizoe, 2003), with varying results. In some cases, retinoid therapy showed efficacy, but sometimes adverse and deleterious effects were observed (Omenn, 2007). The suggestion of retinoids as chemopreventive agents was based on their potential antioxidant properties. However, retinol is better defined as a redox-active molecule, since it may present pro-oxidant activities in vitro (Murata and Kawanishi, 2000) as well as in cell systems (Dal-Pizzol et al., 2000a; Gelain and Moreira, 2007). This may explain the variations observed on retinoid-based therapies in different malignant conditions. We observed here that higher concentrations of retinol (above 20 lM) decreased viability in both SW620 and HT29 cells, which indicates that the deleterious effects observed in some
F. Klamt et al. / Toxicology in Vitro 22 (2008) 873–878
retinoid-based therapies might be caused by excessive retinoid supplementation. In previous works, we observed that treatments with concentrations of retinol up to 5 lM did not had any effect on the modulation of MDR1, MDR2 and MDR3 expression (Frota et al., 2004). Retinol physiologic range for cells is described between 0.2 and 5 lM (Ross, 1993). Also, we previously observed that production of reactive species and oxidative damage are not induced by retinol within this concentration range (Dal-Pizzol et al., 2000a, b; Klamt et al., 2000; 2003). On the other hand, it was observed that retinol 7 lM increases reactive species production and decreases cell viability in different cells (Klamt et al. 2007; Gelain and Moreira, 2007). Considering these previous reports, we used the concentrations of retinol 7 lM in this work, as this concentration was previously observed to modulate MDR in other cells (Frota et al., 2004) and did not decreased viability in both SW620 and HT29 cells (as observed in Fig. 1). Retinoic acid and a synthetic retinoid derivative (6OH-11-O-hydroxyphenanthrene) have been observed to decrease cell proliferation and promote apoptosis in the human colon carcinoma cell LoVo, through a reduction of P-gp synthesis (Bartolini et al., 2006). However, this study did not explore the involvement of reactive species in this effect. Here, we demonstrated that retinol inhibit the expression of MDR1 in the human adenocarcinoma cell line SW620 by a redox-dependent mechanism, as antioxidant treatment inhibited this effect. Redox-mediated inhibition of MDR by retinol was also previously observed in untransformed cells (Frota et al., 2004). Importantly, here we show that MDR1 inhibition by retinol increased SW620 cells susceptibility to VP16 treatment, pointing retinol as a good candidate for an adjuvant agent in antitumorogenic treatment. HT29 cells had no detectable levels of MDR1 expression, and retinol treatment did not change this scenario. This data reinforced our suggestion that the mechanism by which retinol increased susceptibility of SW620 cells to VP16 was MDR1 down-regulation, as this effect was not observed in the non-expressing MDR1 HT29 cells. Besides, the GI50 cytotoxicity assay is frequently used as an index to evaluate affinity of P-gp substrates and inhibitors, as modulators that interact with P-gp and reduce the efflux of the cytotoxic compound will increase the apparent cytotoxicity of the compound tested (Stouch and Gudmundsson, 2002). Considering that retinol increased the cytotoxicity of VP16 to SW60 cells, it is possible that retinol had reduced the efflux of VP16 (and thus increased its efficiency) by inhibiting MDR1 in SW620 cells. Efflux out of cells by P-glycoprotein (P-gp) represents a serious liability for pharmaceuticals, particularly for anticancer drugs. Consequently, the identification of potential substrates or inhibitors of P-gp activity is important to understand their bioavailability and to the development of new strategies to combat cancer MDR phenotype. Retinoids are not substrates for P-gp (Takeshita et al., 2000),
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and we observed here no significant changes in the GI50 values for VP16 in the MDR1 non-expressing HT29 cells. This result suggested that the increase in the toxicity of VP16 in tumor cells pre-treated with retinol was not related to substrate competition, but to the oxidant-mediated inhibition of MDR1 gene expression. Moreover, the development of therapeutic proceedings using vitamin A as an adjuvant in chemotherapy to improve the effects of common anticancer drugs present promising perspectives for the elimination of intrinsic or acquired drug resistance in tumor cells. This, however, requires knowledge of the molecular mechanisms and signal transduction pathways that are involved in the regulation of MDR-related genes by retinol, and the elaboration of sophisticated experimental approaches that efficiently down-regulate the drug transporters. Acknowledgements This work was supported by the Brazilian agencies CNPq, CAPES, FAPERGS and PROPESQ/UFRGS. References Altucci, L., Gronemeyer, H., 2001. Nuclear receptors in cell life and death. Trends in Endrocrinology and Metabolism 12, 460–468. Bartolini, G., Orlandi, M., Papi, A., Ammar, K., Guerra, F., Ferreri, A.M., Rocchi, P.A., 2006. Search for multidrug resistance modulators: the effects of retinoids in human colon carcinoma cells. In Vivo 20 (6A), 729–733. Blomhoff, R., 1994. Vitamin A in Health and Disease. Marcel Dekker, New York. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156–159. Dal-Pizzol, F., Klamt, F., Benfato, M.S., Bernard, E.A., Moreira, J.C.F., 2000a. Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells. Free Radical Research 34, 395–404. Dal-Pizzol, F., Klamt, F., Frota Jr., M.L.C., Moraes, L.F., Moreira, J.C.F., Benfato, M.S., 2000b. Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells. Free Radical Research 33, 677–687. Fenaux, P., Wang, Z.Z., Degos, L., 2007. Treatment of acute promyelocytic leukemia by retinoids. Current Topics in Microbiology and Immunology 313, 101–128. Frota Jr., M.L.C., Klamt, F., Dal-Pizzol, F., Schiengold, M., Moreira, J.C.F., 2004. Retinol-induced MDR1 and MDR3 modulation in cultured rat sertoli cells is attenuated by free radical scavengers. Redox Reports 9, 161–165. Gelain, D.P., Moreira, J.C.F., 2007. Evidence of increased reactive species formation by retinol, but not retinoic acid, in PC12 cells. Toxicology in Vitro, doi:10.1016/j.tiv.2007.11.007. Greenwald, P., Anderson, N., Nelson, S.A., Taylor, P.R., 2006. Clinical trials of vitamin and mineral supplements for cancer chemoprevention. American Journal of Clinical Nutrition 85, 3147S–3317S. Hoyos, B., Imam, A., Korichneva, I., Levi, E., Chua, R., Hammerling, U., 2000. The cysteine-rich regions of the regulatory domains of Raf and Protein Kinase C as retinoid receptor. Journal of Experimental Medicine 192, 835–845. Kakizoe, T., 2003. Chemoprevention of cancer-focusing on clinical trials. Japanese Journal of Clinical Oncology 9, 421–442.
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Klamt, F., Dal-Pizzol, F., Gelain, D.P., Dalmolin, R.S., Birnfeld de Oliveira, R., Bastiani, M., Horn, F., Fonseca Moreira, J.C., 2007. Vitamin A treatment induces apoptosis through an oxidant-dependent activation of the mitochondrial pathway. Cell Biology International, doi:10.1016/j.cellbi.2007.08.018. Klamt, F., Dal-Pizzol, F., Ribeiro, N.C., Bernard, E.A., Benfato, M.S., Moreira, J.C.F., 2000. Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator. Molecular and Cellular Biochemistry 208, 71–76. Klamt, F., Dal-Pizzol, F., Roehrs, R., de Oliveira, R.B., Dalmolin, R., Henriques, J.A., de Andrades, H.H., de Paula Ramos, A.A., Saffi, J., Moreira, J.C.F., 2003. Genotoxicity, recombinogenicity and cellular preneoplasic transformation induced by vitamin A supplementation. Mutation Research 539, 117–125. Krishna, R., Mayer, L.D., 2001. Multidrug resistance (MDR) in cancer mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs. European Journal of Pharmaceutical Sciences 11, 265–283. Murata, M., Kawanishi, S., 2000. Oxidative DNA damage by vitamin A and its derivatives via superoxide generation. Journal of Biological Chemistry 275, 2003–2008. Murphy, L.D., Herzog, C.E., Rudick, J.B., Fojo, A.T., Bates, S.E., 1990. Use of the polymerase chain reaction in the quantitation of MDR1 gene expression. Biochemistry 29, 10351–10356. Njar, V.C., Gediya, L., Purushottamachar, P., Chopra, P., Vasaitis, T.S., Khandelwal, A., Mehta, J., Huynh, C., Belosay, A., Patel, J., 2006. Retinoic acid metabolism blocking agents (RAMBAs) for treatment of cancer and dermatological diseases. Bioorganic and Medicinal Chemistry 14, 4323–4340. Okuno, M., Kojima, S., Matsushima-Nishiwaki, R., Tsurumi, H., Muto, Y., Friedman, S.L., Moriwaki, H., 2004. Retinoids in cancer chemoprevention. Current Cancer Drug Targets 4, 285–298.
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Toxicology in Vitro 22 (2008) 873–878 www.elsevier.com/locate/toxinvit
Inhibition of MDR1 expression by retinol treatment increases sensitivity to etoposide (VP16) in human neoplasic cell line Fa´bio Klamt a,*, Daniel Thompsen Passos b, Mauro Antoˆnio Alves Castro a,b, Daniel Pens Gelain a, Ivana Grivicich b, Jose´ Cla´udio Fonseca Moreira a a
Centro de Estudos em Estresse Oxidativo, Departamento de Bioquı´mica, ICBS/Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ramiro Barcelos 2600 – anexo, Porto Alegre, RS 90035-003, Brazil b Universidade Luterana do Brasil (ULBRA), Canoas, Brazil Received 7 November 2007; accepted 7 January 2008 Available online 15 January 2008
Abstract Multidrug resistance (MDR) is the major obstacle to cancer chemotherapy. MDR phenotype is mainly related to the over-expression of MDR1 gene, being responsible for tumor resistance to several chemotherapeutic drugs. It has been suggested that MDR1 expression is redox-regulated and we have recently described a pro-oxidative effect of retinol. Here we tested the therapeutic use of retinol as a modulator of MDR1 gene expression in tumor cell lines, and verified in situ the enhancement of anticancer drug efficacy. Two human colorectal adenocarcinoma cell lines (HT29, SW620) with different degrees of MDR1 expression were used. Cells were pre-treated with a sublethal dose of retinol and then challenged with the etoposide (VP16) drug. The drug GI50 was assessed by SRB method and levels of MDR1 expression were determined by semi-quantitative rtPCR. Retinol treatment caused a 40% decrease in MDR1 expression and increased VP16 toxicity. MDR1 expression and drug sensitivity were restored to control values when mannitol (a hydroxyl radical scavenger) was co-administrated. Our data point a role to the use of retinol as an adjuvant in the treatment of tumors with MDR phenotype. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Multidrug resistance; Retinol; MDR1; Chemotherapy; Oxidative stress
1. Introduction The resistance of tumor cells to chemotherapeutic interventions remains a major cause of treatment failure in cancer patients. During the past two decades, the research on resistance to cancer chemotherapy was mainly focused on multidrug resistance (MDR). In humans, this phenotype of tumor cells is clinically associated with the over-expression of MDR1 gene and enhancement of drug efflux mediated by P-glycoprotein (P-gp) (Varma et al., 2003). P-gp is a member of the ATP-binding cassette superfamily of transporter proteins, which can export a wide range of chemotherapeutic agents not structurally-related (e.g., vim-
*
Corresponding author. Tel.: +55 51 3308 5577; fax: +55 51 3308 5535. E-mail address: [email protected] (F. Klamt).
0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.01.004
blastine; incristine; doxorubicin; daunomycin; etoposide and others). This action of P-gp contributes to the tumor’s intrinsic or acquired overall resistance against chemotherapy. P-gp expression is positively correlated with drugresistance in cancers of the colon, kidney and adrenal gland (Tsuruo et al., 2003). Besides the great effort to inhibit MDR1 expression in resistant tumor cells, no successful therapeutic proceeding has been developed so far. Moreover, MDR treatment is based on the use of classical inhibitors of P-gp activity (e.g., cyclosporine A; verapamil) but the high incidence of undesired side-effects of these drugs tends to compromise their clinical use (Krishna and Mayer, 2001; Teodori et al., 2002). A more efficient strategy to circumvent the MDR phenotype would be the down-regulation of gene expression coding for the transporters. Recently, it has been pointed out that the expression of P-gp may be
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redox-regulated, which led to new perspectives to the search for new mechanisms to downplay MDR phenotype in cancer cells (Wartenberg et al., 2001). Retinoid supplementation has been widely used therapeutically in the prevention and treatment of several types of diseases, particularly epithelial cancers and leukemia, including both pre-malignant and malignant tumors (Blomhoff, 1994). Retinoid action is classically mediated by specific nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) belonging to the steroid/thyroid superfamily of transcription factors (Altucci and Gronemeyer, 2001). In addition to these classical nuclear-dependent mechanisms, recent works suggested that retinoids may act directly on the modulation of different redox-regulated enzymes, such as Protein Kinase C (PKC) isoforms, serine/threonine kinases, ornithine decarboxilase (ODC) and constitutive Raf (cRaf) (Hoyos et al., 2000; Klamt et al., 2000). Moreover, a growing body of evidence has demonstrated that b-carotenoids, vitamin A and retinoids have pro-oxidant properties (Klamt et al., 2003; Dal-Pizzol et al., 2000a,b; Murata and Kawanishi, 2000). Retinol causes an increase in 8-oxo,dGuanosine adducts in the DNA of treated cells, suggesting the generation of high reactive hydroxyl radical (OH) by retinol treatment (Dal-Pizzol et al., 2000a). As demonstrated, once formed, free radicals can act as cellular second messengers, regulating several signal transduction pathways and consequently, gene expression (Frota et al., 2004). The experiments presented in this report were designed to investigate both (1) if retinol supplementation is able to modulate MDR1 gene expression and (2) if modulation of MDR1 causes an increase in anticancer drug efficacy. Our results provide evidence for the involvement of free radical generation as a possible mechanism of retinol action, as co-treatment with mannitol restored MDR1 expression and drug sensitivity to control values. The data are discussed with reference to the use of retinol supplementation as an adjuvant during cancer chemotherapeutic interventions.
by sulforhodamine B (SRB) method, as described in ‘‘Cell Growth Inhibition Measurements”, and MDR expression was assessed by semi-quantitative rtPCR, as described below. The dose and time exposure of retinol chosen (7 lM, 24 h) were based on the following previous observations: (i) this concentration increases free radicals production and activates redox signaling in different experimental models (Klamt et al., 2000; Dal-Pizzol et al. 2000b; Klamt et al., 2003); (ii) it was the concentration observed to abolish MDR1 and MDR3 gene expression in cultured rat Sertoli cells (Frota et al., 2004); and (iii) data depicted at Fig. 1 (results section) showed that these conditions did not induce cytotoxicity in SW620 and HT29 cell lines. 2.2. Cell growth inhibition measurements
2. Materials and methods
Cells were separated into a single-cell suspension in culture medium by trypsinization and seeded into 96-well culture plates at a density of 7.5 103 cells/cm2 (initial cell density). Cell growth was performed at 37 °C in a 5% CO2 humidified atmosphere. The medium was replenished each 48 h after seeding and the cell number was determined periodically by the sulforhodamine B (SRB) staining assay. Briefly, adherent cell cultures were fixed in situ by adding 50 ll of cold 50% (w/v) trichloroacetic acid (TCA) (final concentration, 10% TCA) for 60 min at 4 °C. The supernatant was then discarded, and the plates were washed five times with deionized water and dried. One hundred microlitres of SRB solution (0.4% in 1% acetic acid, w/v) was added to each microtitre well and the culture was incubated for 10 min at room temperature (25 °C). Unbound SRB was removed by washing five times with 1% acetic acid and the plates were air dried. The bond stain was solubilized with Tris buffer, and the absorbance was read on an automated spectrophotometric plate reader at a single wavelength of 515 nm. Cell growth was assessed in the SRB-staining assay by whole culture protein determination, showing sensitivity and reproducibility. The GI50 (growth inhibition index) value was obtained by determining the concentration of retinol that inhibited cell growth by 50% using this method.
2.1. Cell cultures and treatments
2.3. RNA extraction and PCR
The human colon adenocarcinoma cell lines SW620 and HT29, which have different degrees of MDR1 ABCB1; ATP-binding cassette, sub-family B member (1) gene expression were obtained from the American Type Culture Collection (ATCC). Basal levels of MDR1 gene expression were obtained based on the analysis of the cDNA microarray gene expression Serial Analysis of Gene Expression (SAGE) databank from the NCI’s Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/SAGE) (Fig. 1A). Cells in the exponential growth phase were routinely cultured, pre-treated with retinol 7 lM by 24 h and then exposed to different concentrations of etoposide (VP16) for additional 72 h. Cell growth inhibition was assessed
Total cellular RNA was isolated by the guanidinium isothiocyanate/phenol–chloroform method (Chomczynski and Sacchi, 1987). In each experiment, water was used as a negative control for contamination. Reverse transcription of 1 lg of the total RNA using 10 pmol of specific 30 primer was performed for 50 min at 42 °C using 20 lL of reverse transcriptase buffer (Gibco/BRL) containing 1 mM each of dATP, dGTP, dCTP, and dTTP, and 200 U of AMV reverse transcriptase (Gibco–BRL), as described (Murphy et al., 1990). The samples were then heated at 95 °C for 5 min to terminate the reverse transcription. The resulting cDNA was serially diluted in water from 1:2 (500 ng of RNA) to 1:32 (31.25 ng of RNA). To these dilutions were
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Fig. 1. (A) Basal MDR expression levels were examined in HT29 and SW620 cells by meta-analysis of cDNA microarray gene expression SAGE databank from the NCI’s Cancer Genome Anatomy Project. (B) GI50 values of retinol on both cell lines were calculated as described in Section 2. SW620 cells were treated with retinol 7 lM or retinol plus mannitol 1 mM for 24 h and MDR1 expression levels were evaluated by semi-quantitative rtPCR (C and D). Results are expressed as mean ± SEM. * Significantly different from respective control (p < .05).
added 50 lL of PCR buffer (Promega) containing 200 lM dNTP, 2.5 lL of the cDNA template, 1.25 U of Taq DNA polymerase (Perkin–Elmer Cetus), 1 lM of MDR1 primers, and 0.5 lM of b-actin primers. Primers were selected from two exons separated by one or more long intronic sequences, which allowed identification of contaminating genomic DNA. The sequences of primers used were as following: MDR1 (PCR product = 157 bp) 50 -CCCATCATTgCAATAgCAgg-30 – forward and 50 -gTTCAAACTTCTgCTCCTgA-30 – reverse; b-actin (PCR product = 228) 50 -CgggAAATCgTgCgTgACAT-30 – forward and 50 -ggAgTTgAAggTAgTTTCgTg-30 – reverse). The reaction mixture was overlaid with 100 lL of mineral oil, and heated at 95 °C for 2 min and carried out for 30 cycles. One cycle consisted of denaturation at 94 °C for 1 min, 15 s; annealing at 55 °C for 1 min, 15 s; and extension at 72 °C for 2 min. The PCR products were subjected to electrophoresis in 1X Tris-borate/2 mM EDTA buffer though 2% agarose containing ethidium bromide for UV visualization. 2.4. Data analysis PCR products intensities were analyzed by scanning densitometry (ImageJ 1.36b; NIH, USA), using b-actin as housekeeper gene control. The results were plotted as densitometric arbitrary units against the quantity of RNA present in the PCR reaction diluted from the reverse transcriptase reaction. For GI50 drug values, the results were
expressed as the mean ± SEM. Data were analyzed by one-way analysis of variance (ANOVA), using a Neuman–Keuls test to compare means values across groups. Differences were considered to be significant when p < .05. 3. Results Meta-analysis of cDNA microarray gene expression revealed that MDR1 expression values in SW620 cells are significantly increased compared to HT29 cells (Fig 1A). This data indicated that these two cell lines are suitable to compare the effects of chemotherapeutic drugs on different human colon adenocarcinoma cell lines, one presenting low MDR1 expression (HT29) and the other with high expression levels of MDR1 (SW620). Next, we evaluated the GI50 values for HT29 and SW620 cells treated with varying concentrations of retinol (up to 40 lM). With concentrations up to 10 lM, retinol caused no statistically detectable growth inhibition in both cell lines (Fig. 1B). However, retinol 20 lM induced a significant decrease (40%) in SW620 cell number, with a further decrease observed at 40 lM. The number of HT29 cells was only decreased by retinol at 40 lM (Fig. 1B). These results indicated that concentrations up to 10 lM of retinol do not affect cell viability. Next, we evaluated the effect of retinol on MDR1 gene expression in both cell lines. As expected, semi-quantitative rtPCR analysis did not found detectable MDR1 expression levels in HT29 control cells, and retinol did not change this
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Fig. 2. HT29 (A) and SW620 (B) cells pre-treated with retinol 7 lM or retinol plus mannitol 1 mM were subjected to VP16 treatment and cell viability was evaluated. Results are expressed as percentage of control (mean ± SEM). * Significantly different from respective control (p < .05).
profile (not shown). In the MDR1-expressing SW620 cells, however, retinol incubation significantly decreased MDR1 expression to levels approximately 50% of control cells (Fig. 1C and D). To investigate if MDR1 inhibition by retinol was mediated by a redox-dependent mechanism, we evaluated the effect of the antioxidant mannitol, a classical hydroxyl radical scavenger, on the effect of retinol upon MDR1 expression in SW620 cells. Mannitol co-administration restored MDR1 gene expression to control values (Fig. 1C and D); ethanol (the retinol vehicle), or mannitol alone, had no effect on the degree of MDR1 gene expression (data not shown). These results indicated that MDR1 down-regulation by retinol in SW620 cells is mediated by a redox-dependent mechanism. Next, to assess if the retinol-mediated decrease in MDR1 gene expression cause an increase in the cellular retention of anticancer drug, we determined the GI50 values of the anticancer compound etoposide (VP16, which is a classical P-gp substrate) in HT29 and SW620 cells pre-treated with retinol. VP16 concentration curves (0–20 lM) showed no differences on inhibition of cell growth between untreated and retinol pre-treated HT29 cells, and addition of the antioxidant mannitol to the pre-treatment also presented no effect (Fig. 2A). In contrast, retinol pre-treated SW620 cells were more susceptible to the inhibition effect of increasing concentrations of VP16 (Fig. 2B); the antioxidant co-treatment with mannitol reversed the effect of retinol, indicating the involvement of a redox-dependent mechanism on this effect. Calculated GI50 values of VP16 on both cell lines pre-treated with retinol are depicted at Table 1. Since these cells express high levels of MDR1, and this expression is inhibited by retinol in a redox-dependent way (Fig. 1), this result indicates that retinol pre-treatment increases SW620 susceptibility to VP16 through an oxidant down-regulation of MDR1. 4. Discussion Retinoid-based therapies present potential perspectives for the treatment of different types of diseases related to cell
Table 1 GI50 (lM) values for etoposide (VP16) in human neoplasic cell lines exposed for 72 h, pre-treated with 7 lM of retinol Treatments
Cell lines HT29
SW620
VP16 VP16 pre-treated with retinol VP16 pre-treated with retinol + mannitol
1.15 ± 0.23 0.93 ± 0.14 1.08 ± 0.11
0.64 ± 0.12 0.20 ± 0.07a 0.57 ± 0.18
Two human adenocarcinoma cell lines (ATCC) were selected by different degree in MDR1 expression, and cultivated as described in ‘‘Materials and Methods” section. HT29 (with undetected MDR1 gene expression) and SW620 human adenocarcinoma cell line (with higher level of MDR1 expression) were pre-treated with 7 lM of retinol for 24 h, washed, exposed to different concentrations of VP16, and the GI50 of the drug was determined. Data represent means ± SD of three independent experiments carried out in triplicates. a Different from VP16-treated cells and from VP16 pre-treated with retinol + mannitol cells (p < .01).
cycle disruption/cell death and increased ROS formation. Diseases such as skin cancer, lung cancer, Parkinson’s disease and Alzheimer disease, among others, were suggested to be treatable by retinoid-based therapies (Njar et al., 2006; Greenwald et al., 2006; Okuno et al., 2004; Ono and Yamada, 2007). Protocols for treatment of malignant diseases such as leukemia and lung cancer have been proposed and clinical trials have been carried out (Fenaux et al., 2007; Kakizoe, 2003), with varying results. In some cases, retinoid therapy showed efficacy, but sometimes adverse and deleterious effects were observed (Omenn, 2007). The suggestion of retinoids as chemopreventive agents was based on their potential antioxidant properties. However, retinol is better defined as a redox-active molecule, since it may present pro-oxidant activities in vitro (Murata and Kawanishi, 2000) as well as in cell systems (Dal-Pizzol et al., 2000a; Gelain and Moreira, 2007). This may explain the variations observed on retinoid-based therapies in different malignant conditions. We observed here that higher concentrations of retinol (above 20 lM) decreased viability in both SW620 and HT29 cells, which indicates that the deleterious effects observed in some
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retinoid-based therapies might be caused by excessive retinoid supplementation. In previous works, we observed that treatments with concentrations of retinol up to 5 lM did not had any effect on the modulation of MDR1, MDR2 and MDR3 expression (Frota et al., 2004). Retinol physiologic range for cells is described between 0.2 and 5 lM (Ross, 1993). Also, we previously observed that production of reactive species and oxidative damage are not induced by retinol within this concentration range (Dal-Pizzol et al., 2000a, b; Klamt et al., 2000; 2003). On the other hand, it was observed that retinol 7 lM increases reactive species production and decreases cell viability in different cells (Klamt et al. 2007; Gelain and Moreira, 2007). Considering these previous reports, we used the concentrations of retinol 7 lM in this work, as this concentration was previously observed to modulate MDR in other cells (Frota et al., 2004) and did not decreased viability in both SW620 and HT29 cells (as observed in Fig. 1). Retinoic acid and a synthetic retinoid derivative (6OH-11-O-hydroxyphenanthrene) have been observed to decrease cell proliferation and promote apoptosis in the human colon carcinoma cell LoVo, through a reduction of P-gp synthesis (Bartolini et al., 2006). However, this study did not explore the involvement of reactive species in this effect. Here, we demonstrated that retinol inhibit the expression of MDR1 in the human adenocarcinoma cell line SW620 by a redox-dependent mechanism, as antioxidant treatment inhibited this effect. Redox-mediated inhibition of MDR by retinol was also previously observed in untransformed cells (Frota et al., 2004). Importantly, here we show that MDR1 inhibition by retinol increased SW620 cells susceptibility to VP16 treatment, pointing retinol as a good candidate for an adjuvant agent in antitumorogenic treatment. HT29 cells had no detectable levels of MDR1 expression, and retinol treatment did not change this scenario. This data reinforced our suggestion that the mechanism by which retinol increased susceptibility of SW620 cells to VP16 was MDR1 down-regulation, as this effect was not observed in the non-expressing MDR1 HT29 cells. Besides, the GI50 cytotoxicity assay is frequently used as an index to evaluate affinity of P-gp substrates and inhibitors, as modulators that interact with P-gp and reduce the efflux of the cytotoxic compound will increase the apparent cytotoxicity of the compound tested (Stouch and Gudmundsson, 2002). Considering that retinol increased the cytotoxicity of VP16 to SW60 cells, it is possible that retinol had reduced the efflux of VP16 (and thus increased its efficiency) by inhibiting MDR1 in SW620 cells. Efflux out of cells by P-glycoprotein (P-gp) represents a serious liability for pharmaceuticals, particularly for anticancer drugs. Consequently, the identification of potential substrates or inhibitors of P-gp activity is important to understand their bioavailability and to the development of new strategies to combat cancer MDR phenotype. Retinoids are not substrates for P-gp (Takeshita et al., 2000),
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and we observed here no significant changes in the GI50 values for VP16 in the MDR1 non-expressing HT29 cells. This result suggested that the increase in the toxicity of VP16 in tumor cells pre-treated with retinol was not related to substrate competition, but to the oxidant-mediated inhibition of MDR1 gene expression. Moreover, the development of therapeutic proceedings using vitamin A as an adjuvant in chemotherapy to improve the effects of common anticancer drugs present promising perspectives for the elimination of intrinsic or acquired drug resistance in tumor cells. This, however, requires knowledge of the molecular mechanisms and signal transduction pathways that are involved in the regulation of MDR-related genes by retinol, and the elaboration of sophisticated experimental approaches that efficiently down-regulate the drug transporters. Acknowledgements This work was supported by the Brazilian agencies CNPq, CAPES, FAPERGS and PROPESQ/UFRGS. References Altucci, L., Gronemeyer, H., 2001. Nuclear receptors in cell life and death. Trends in Endrocrinology and Metabolism 12, 460–468. Bartolini, G., Orlandi, M., Papi, A., Ammar, K., Guerra, F., Ferreri, A.M., Rocchi, P.A., 2006. Search for multidrug resistance modulators: the effects of retinoids in human colon carcinoma cells. In Vivo 20 (6A), 729–733. Blomhoff, R., 1994. Vitamin A in Health and Disease. Marcel Dekker, New York. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156–159. Dal-Pizzol, F., Klamt, F., Benfato, M.S., Bernard, E.A., Moreira, J.C.F., 2000a. Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells. Free Radical Research 34, 395–404. Dal-Pizzol, F., Klamt, F., Frota Jr., M.L.C., Moraes, L.F., Moreira, J.C.F., Benfato, M.S., 2000b. Retinol supplementation induces DNA damage and modulates iron turnover in rat Sertoli cells. Free Radical Research 33, 677–687. Fenaux, P., Wang, Z.Z., Degos, L., 2007. Treatment of acute promyelocytic leukemia by retinoids. Current Topics in Microbiology and Immunology 313, 101–128. Frota Jr., M.L.C., Klamt, F., Dal-Pizzol, F., Schiengold, M., Moreira, J.C.F., 2004. Retinol-induced MDR1 and MDR3 modulation in cultured rat sertoli cells is attenuated by free radical scavengers. Redox Reports 9, 161–165. Gelain, D.P., Moreira, J.C.F., 2007. Evidence of increased reactive species formation by retinol, but not retinoic acid, in PC12 cells. Toxicology in Vitro, doi:10.1016/j.tiv.2007.11.007. Greenwald, P., Anderson, N., Nelson, S.A., Taylor, P.R., 2006. Clinical trials of vitamin and mineral supplements for cancer chemoprevention. American Journal of Clinical Nutrition 85, 3147S–3317S. Hoyos, B., Imam, A., Korichneva, I., Levi, E., Chua, R., Hammerling, U., 2000. The cysteine-rich regions of the regulatory domains of Raf and Protein Kinase C as retinoid receptor. Journal of Experimental Medicine 192, 835–845. Kakizoe, T., 2003. Chemoprevention of cancer-focusing on clinical trials. Japanese Journal of Clinical Oncology 9, 421–442.
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