Control of Multidrug Resistance Gene mdr1 and Cancer Resistance to Chemotherapy by the Longevity Gene sirt1

Cellular senescence, traditionally associated with organismal aging, has emerged recently as a key determinant of cancer chemotherapy outcome (1–5). Our laboratory and others have shown that the stress level required for induction of senescence is significantly lower than those necessitated for other cellular toxic responses, such as apoptosis or necrosis (6–8), and that forcing cancer cells to undergo senescence was sufficient for reversal of their resistance phenotype (9). These findings suggested that senescence deficiency may regulate the early events that control development of resistance to chemotherapy. Thus, identification and targeting of antisenescence genes may have potential therapeutic utility for the prediction and/or prevention of drug resistance. In an effort to identify such genes, we directed our focus to research in the field of aging. Recently, enhanced activity of the silent information regulator (sir2) gene was found to be associated with increased longevity in yeast (10, 11), worm (12), flies (13), and rodents (14). Its human homologue, sirt1, was also described to be associated with longevity and was believed to act primarily by inhibiting cellular senescence (15–19). This later characteristic of sirt1 prompted us to investigate its potential role in mediating resistance to chemotherapy in cancer. Our results indicate that this gene was overexpressed in all the drug-resistant cell lines tested in our laboratory as well as in tumor specimens from cancer patients treated with chemotherapy. Moreover, a positive regulatory relationship was identified between sirt1 and the multidrug resistance gene mdr1, suggesting that this longevity gene was not only associated with but also plays an active role in this phenomenon.

Cell lines, biopsies, and reagents. Human neuroblastoma SKN-SH, osteosarcoma SaOS2, breast cancer MCF7, and 293 cell lines were purchased from American Type Culture Collection (Rockville, MA). Drug-resistant cells were generated by continuous incubation of parental cell lines with stepwise increases in drug concentration over a period of 3 to 6 months. The ovarian cancer cell lines A2780 and IGROV1 and corresponding cisplatin-resistant cells were a generous gift from Dr. Mary C. Hendrix. Tumor biopsies were obtained at surgery and frozen at −70°C until use. DMEM and fetal bovine serum (FBS) were obtained from BioWhittaker (Walkersville, MD). The following drugs and reagents were obtained from the companies cited: doxorubicin, etoposide, and cisplatin (Sigma, St. Louis, MO); antibody to Sirt1 (Novus Biological, Littleton, CO); antibody to β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA); secondary antibodies conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA); enhanced chemiluminescence reagents (ECL, Amersham, Arlington Heights, IL); and Immobilon-P transfer membrane for Western blots (Millipore, Bedford, MA).

Western blot. Cells were seeded in DMEM containing 10% FBS, cultivated for 48 hours, then lysed in a lysis buffer [50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 100 mmol/L NaF, 1 mmol/L MgCl₂, 1.5 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 μg/mL leupeptin, 1 mmol/L phenyl-methyl-sulfonyl-fluoride]. Tumor biopsies were homogenized in the lysis buffer described above and centrifuged to remove insoluble material. Equal quantities of protein were separated by electrophoresis on a 12% SDS-PAGE gel and transferred onto Immobilon-P membranes. Proteins of interest were identified by reaction with specific primary and secondary antibodies linked to horseradish peroxidase. Reactive bands were detected by chemiluminescence.

Reverse transcription-PCR. RNA extraction and reverse transcription-PCR (RT-PCR) reactions were done using the GeneAmp RNA PCR kit (part no. N808-0017, Applied Biosystems, Foster City, CA) according to the procedure of the manufacturer. PCR products were visualized on 2% agarose gel with 10% ethidium bromide.

Small interfering RNA design and transfection. The human Sirt1 small interfering RNA (siRNA; ref. 14) was synthesized by Dharmacon (Lafayette, CO). On the day before transfection, 3 × 10⁵ doxorubicin-resistant osteosarcoma cells were seeded into six-well plates and grown in 2.5 mL of DMEM supplemented with 10% FBS. After 24 hours in culture, 25 μL of 20 μmol/L stock solution of siRNA duplexes were transfected into cells using GeneSilencer siRNA Transfection Reagent kit according to the protocol of the manufacturer (Gene Therapy Systems, San Diego, CA). After 24 hours of incubation, cells were treated with doxorubicin and maintained in culture for an additional 48 hours before measuring cell viability and expression of the silenced molecules with Western blot or RT-PCR.

Assay of the mdr1 promoter activity. Cells were cotransfected either with Sirt1 siRNA or the plasmid PGL3 containing the promoter sequence for the mdr1 gene (20). After 48 hours of incubation, cells were lysed and the luciferase activity measured using the Luciferase Assay System kit (Promega, Madison, WI).

Transfection with sirt1. sirt1 transfectants were produced using 293 cells maintained in DMEM medium supplemented with 10% FCS in six-well plates. The pYSIR2 plasmid (a generous gift from Dr. R. Weinberg; ref. 17) was introduced alone or in combination with the PGL3 plasmid containing the mdr1 promoter region using LipofectAMINE Reagent 2000 following the protocol of the manufacturer (Invitrogen, Carlsbad, CA). After 48 hours, expressions of sirt1 and mdr1 were detected by RT-PCR. Expressions of these two genes at the protein level were determined by Western blot. Activation of the mdr1 promoter was assayed as described above.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells were incubated with doxorubicin at concentrations varying between 1 × 10⁻⁹ and 1 × 10⁻⁶ mol/L for 96 hours. Viable cells were quantitatively estimated by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT (10 μL of 5 mg/mL solution) was added to each well of the titration plate and incubated for 4 hours at 37°C. The cells were then solubilized by the addition of 100 μL of 10% SDS/0.01 mol/L HCl and incubated for 15 hours at 37°C. The absorbance of each well was determined in an ELISA plate reader using an activation wavelength of 570 nm and a reference wavelength of 650 nm. The percentage of viable cells was determined by comparison with untreated control cells.

To establish whether Sirt1 could be associated with development of drug resistance in cancer, we used various drug-sensitive and drug-resistant cancer cell lines representing neuroblastoma, osteosarcoma, mammary, and ovarian cancinomas (9). As shown in Fig. 1A, Western blot analysis using a polyclonal antibody to this molecule indicated that its expression was up-regulated in all drug-resistant cells tested when compared with their drug-sensitive counterparts. The validity of this observation seemed to be independent of cell type or the nature of drugs used to generate resistant cells, suggesting that increased Sirt1 expression may represent a general phenomenon associated with resistance to chemotherapy. To verify whether Sirt1 could be associated with treatment response in cancer patients, matching biopsy specimens obtained at either diagnosis or after chemotherapy were analyzed by Western blot (Fig. 1B). Although the number of specimen studied was limited (n = 4), the data clearly indicate that elevated amounts of Sirt1 were invariably detected following treatment with chemotherapeutic agents. To verify or rule out the possibility that increased Sirt1 amounts occurred at the gene expression or posttranslational level, pairs of drug-sensitive and drug-resistant cell lines (Fig. 1C) and biopsy specimens from untreated and drug-treated patients (Fig. 1D) were analyzed for expression of this molecule by RT-PCR. The results were similar to those obtained by Western blot using a polyclonal antibody (Fig. 1A and B), suggesting that changes in the amount of Sirt1 occurred at the gene expression level.

Sirt1 belongs to a sirtuin family of seven members (21); therefore, we investigated the possibility that other sirtuins may also be associated with drug resistance. The analysis of RT-PCR profiles in IGROV1 cells and their cisplatin-resistant counterpart as well as in specimen from patient #3 (Fig. 1E) revealed that except for Sirt1, no other sirtuin seemed to be affected by exposure of cancer cells to therapeutic agents in vitro or in vivo. These findings suggest that Sirt1 may represent a stress-responsive molecule and, with regards to chemotherapy, this molecule may serve as marker for prognosis.

To determine the stress level and time required for Sirt1 expression, neuroblastoma cells, SKN-SH, were subjected to treatment with 0.5 × 10⁻⁸ mol/L doxorubicin, which inhibits proliferation, or 10⁻⁶ mol/L, which induces apoptosis in these cells (8), and expression of this molecule was measured over a period of 96 hours. The results (Fig. 2A) show that increased amounts of Sirt1 were observed at low, but not at high, drug concentrations, suggesting that only the stress levels that inhibit proliferation may activate the Sirt1 pathway. This cellular response was quite rapid because increased amounts of Sirt1 were detected as early as 24 hours of doxorubicin treatment and culminated at 48 hours of incubation. Sirt1 expression in response to drug treatment was also transient and after 72 hours of exposure, a noticeable decrease was observed, perhaps due to reduced drug activity and/or availability. Of particular interest is the correlation of expression of Sirt1 with that of the cell cycle inhibitor p21/WAF1 but not with caspase-3 activation (Fig. 2A), which suggests that Sirt1 may exert its protective function only in response to sublethal stress levels. A similar relationship was observed in the ovarian cancer cell line A2780 treated with cisplatin (Fig. 2B) and, as in the neuroblastoma cell line, there was also an inverse correlation between the activation of caspase 3 and expressions of Sirt1 and p21/WAF1. This puzzling observation prompted us to hypothesize that Sirt1 may be a substrate for caspase 3. Indeed, we have found that when recombinant Sirt1 was incubated with purified caspase 3, its degradation could be detected as early as 30 minutes (Fig. 2C). Together, these findings indicate that Sirt1 is expressed in response to antiproliferative stress levels; however, its protective function may be lost if cells are exposed to apoptosis-inducing stimuli. The relationship between Sirt1 and p21/WAF1 seems to be in favor of the view that Sirt1 may exert its protective function by signaling for a reduction in proliferation rate to allow cellular adaptation and survival in a cytotoxic environment.

To gain further insight into the mechanisms that govern drug resistance through Sirt1, we investigated the possibility that this molecule may regulate, directly or indirectly, the expression and/or activity of genes known to be implicated in drug resistance. The most widely described among these are the drug efflux pump P-glycoprotein (22), which is encoded by the mdr1 gene, superoxide dismutase (SOD) (23, 24), and glutathione S-transferase-π (GST-π), which catalyzes drug inactivation (25). We used siRNAs specific for Sirt1 to knockdown the expression of this molecule in drug-resistant cells and to determine whether this affects expression of these genes. After transfection, the amount of Sirt1 was markedly reduced (Fig. 3A); in addition, sensitivity to doxorubicin increased in the transfected cells when compared with their nontransfected or vehicle-treated counterparts (Fig. 3B). We have shown previously that a 1 μmol/L drug concentration leads to the death of 80% of drug-sensitive SaOS2 cells and only 20% of their drug-resistant counter parts (9). Sirt1 siRNA enhances cellular response in the drug-resistant cells by an additional 25% to 30%, resulting in a total cell death of 45% to 50%. Although this is not a complete reversal of drug resistance, it represents a significant enhancement of cellular response to the drug.

These findings indicate that Sirt1 is not only associated with but may also play an active role in the development of resistance to drugs. Significantly, siRNA-mediated down-regulation of Sirt1 was associated with decreased expression of P-glycoprotein but it did not affect the expression of SOD or GST-π (Fig. 3C). Similar findings were obtained by RT-PCR (Fig. 3D), indicating that regulation of P-glycoprotein expression by Sirt1 occurred at the transcriptional level rather than at the posttranslational level. Further confirmation of the modulatory action of Sirt1 on P-glycoprotein expression is illustrated by the reduction in mdr1 promoter activity on cotransfection with Sirt1 siRNA (Fig. 3E). Cellular proliferation was not significantly altered during the time of the experiment (data not shown), suggesting that the effect of Sirt1 siRNA on the activity of mdr1 promoter was specific and not due to difference in growth rate.

Gain of Sirt1 function was also investigated to determine whether it may influence mdr1 expression, and for this we used two approaches: First, we used resveratrol, a phenol contained in red wine that has been shown to induce Sirt1 activity and stimulate its protective function (26, 27). The data indicate that on cellular exposure to this compound for 48 hours, mdr1 gene expression increased relatively compared with untreated cells as measured by semiquantitative PCR (Fig. 4A) and, as expected, the activity of the corresponding promoter was also significantly enhanced (Fig. 4B). In the second approach, ectopic overexpression of the sirt1 gene was carried out in 293 cells and, as expected, this was accompanied with a strong expression of the mdr1 gene (Fig. 4C). Activity of the mdr1 promoter was also increased (Fig. 4D), as was the induction of P-glycoprotein expression (Fig. 4E), in the Sirt1-transfected cells. More importantly, cells stably transfected with the Sirt1 gene displayed resistance to doxorubicin (Supplementary data 1). Taken together, these findings provide conclusive evidence that expression of the mdr1 gene can be controlled by the histone deacetylase Sirt1 and highlight the importance of chromatin remodeling in the regulation of drug resistance development.

In conclusion, the present study has uncovered a novel function of the longevity molecule Sirt1 as a potential marker and modulator of the drug resistance phenotype in cancer. As this molecule seemed to control the expression of a major drug resistance molecule, P-glycoprotein, new avenues may be open for further understanding of the causes leading to the onset of drug resistance. With regards to the relationship between aging and cancer, the present study sheds light on common signaling pathways that may be used by somatic and cancer cells to overcome toxic stress.

Grant support: National Cancer Institute NIH grant 1R01 CA096616-01A1, John W. Anderson Foundation, North Suburban Medical Research Junior Board, Medical Research Junior Board Foundation, Medical Research Institute Council, and R. Wile Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Mary C. Hendrix (Children's Memorial Research Center, Northwestern University, Chicago, IL) for generously proving drug-sensitive and drug-resistant ovarian cancer cells, Dr. Brannimir I. Sikic (Stanford University, Palo Alto, CA) and Kevin G. Chen (NIH, Bethesda, MD) for providing the mdr1 construct, and Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Massachussett Institute of Technology, Cambridge, MA) for the pYSIR2 construct.