Cellular resistance to cisplatin is multifactorial and may consist of mechanisms that limit cisplatin uptake and accumulation, altered repair mechanisms, and changes that promote cell survival (20
). Our previous study and the work of others has shown that altered uptake of cisplatin occurs because of reduced membrane-binding/transport proteins and reduced endocytosis (9
). In this work, we show that SIRT1 upregulation also contributes to cisplatin resistance in association with altered mitochondrial metabolism.
It is known that cells derive up to 95% of their energy through oxidative phosphorylation carried out in mitochondria. Subtle alterations in the glycolytic metabolic pathway could affect energy biosynthetic reactions and cellular susceptibility in the face of stress. Harper et al. identified and characterized a cellular metabolic strategy that differentiates drug-resistant cells from drug-sensitive cells (22
). According to this strategy, drug-resistant cells use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limiting, and mitochondria become dysfunctional in resistant cells. Abnormal mitochondrial structure may have substantial impact on cellular metabolism, and especially on mitochondrial metabolism, which plays an important role in stress resistance, chromatin-dependent gene regulation, and genome stability. Support for mitochondria as a direct target of cisplatin toxicity comes from studies which show that intestinal epithelial IEC-6 rho(0) cells with reduced number of mitochondria are more resistant to cisplatin than normal cells (23
). This is consistent with our results, which showed that uptake of 2-deoxyglucose was reduced due to dysfunction and altered morphology of mitochondria in Cp-r cells.
In addition, glucose transporter 1 (GluT1), a common molecular target of most anti-diabetic drugs, is no longer localized to the plasma membrane in CP-r cells (data not shown), which is similar to the recycling defect we have previously reported for a transmembrane protein (multidrug resistant protein 1, MRP1) in malignant CP-r cells (5
). GluT1, the mediator of basal glucose uptake, is the primary glucose transporter in human cancer and is expressed by most cancer cells. Reduced 2-deoxyglucose uptake in CP-r cells is likely due to the altered localization of GluT1, the main membrane protein transporting glucose into tumor cells, by a mechanism not yet elucidated. Aft et al. found that overexpression of Glut1 transporter protein was associated with increased glucose uptake in breast cancer cells treated with 2-deoxyglucose (24
). The disruption of glucose uptake in CP-r cells is associated with abnormal mitochondrial function, as measured by oxygen consumption and mitochondrial membrane potential. It was found previously that SIRT1 overexpression could reduce oxygen consumption and alter mitochondrial bioenergenesis by acting with PGC-1 in PC-12 cells (19
). Given that oxygen consumption is linked to the generation of reactive oxygen species and reactive oxygen species levels correlate with cisplatin toxicity (25
), these results may have important implications for how SIRT1 regulates cisplatin resistance.
Limitation of glucose utilization alone results in SIRT1 overexpression in KB-3-1 and BEL7404 cells, and increases the resistance of parental CP-s KB-3-1 and BEL7404 cells to cisplatin. The bioenergetic dysfunction in CP-r KB-CP 20 and BEL7404-CP 20 cells suggests that reducing glucose metabolism also leads to overexpression of SIRT1, which confers cisplatin resistance to KB-3-1 and BEL7404 cells. How might SIRT1 overexpression contribute to cisplatin resistance? Cisplatin perturbs nucleic acid structure and function when it incorporates into DNA or RNA, thereby causing tumor cells to be killed. SIRT1 is known to deacetylate p53 protein, which recognizes and binds to DNA modified with cisplatin (26
). It has been proposed that deacetylation by SIRT1 reduces p53-dependent apoptosis in response to DNA damage (27
). Interactions between p53 and SIRT1 after platination might provide a molecular link between DNA damage and p53-mediated DNA repair. The cross-resistance of the CP-r cells studied here to numerous cytotoxic agents, including heavy metals, alkylating agents, and methotrexate (5
) could reflect such a general effect on apoptosis mediated by SIRT1.
It is well known that SIRT1 is involved in cellular metabolism, and in order to function, SIRT1 requires the presence of NAD+
. However, there is an active debate about whether NAD+
directly controls in vivo
activity of SIRT1 (28
). In this study, we found that NAD+
concentration was higher in CP-r cells that overexpress SIRT1 (). If the cellular NAD+
concentration were low, SIRT1 deacetylase activity could be attenuated, thus increasing the chances of a cell becoming senescent or apoptotic through the acetylated form of p53 (30
). Lai and colleagues (31
) provide evidence of this, by demonstrating increased acetylation of wild-type p53 during cisplatin-induced apoptosis. In addition, the requirement for NAD+
in the deacetylase activity of SIRT1 suggests that SIRT1 might be involved in metabolism of NAD+
as a metabolic regulator. SIRT1 may function as a bridge, coordinating metabolic status with regulation of key target genes involved in cancer resistance to cisplatin. Thus, higher NAD+
and SIRT1 levels seen in CP-r cells would favor a less effective apoptotic pathway.
Processes that require energy, such as fatty acid synthesis, protein synthesis, and cell growth are curtailed in CP-r cells (9
), possibly through the effects of SIRT1 as a central modulator. Overexpressed SIRT1 in CP-r cells might result in a higher threshold for apoptosis by targeting numerous cellular factors. It is possible that SIRT1 acts as a metabolic sensor, via its NAD+
dependence, that links energy consumption to a transcriptional program that modulates response to stress. Deacetylation of transcriptional complexes by SIRT1 is generally associated with diminished transcriptional activation by removing the acetyl group from lysines of certain transcription factors (e.g. NF-κB, FOXO and p53, etc.) (8
). Several transcription factors, such as NF-κB, YB-1, mtTFA, Ets-1 and AP-1 are activated by cisplatin treatment, and are involved in drug resistance and DNA repair (34
). SIRT1 may act as a scaffold to tether various transcriptional complexes. Brunet et al. (8
) found that SIRT1 conferred resistance to etoposide by augmenting DNA repair, and thymocytes derived from SIRT1 knockout mice were found to be more sensitive to ionizing radiation. Therefore, overexpression of SIRT1 might shift the transcription-dependent response of cisplatin resistance away from cell death and toward cell survival.
Cellular mechanisms of resistance to cisplatin are multifactorial and contribute to severe limitation in the use of cisplatin in the clinic. Our research shows that cisplatin resistance reflects a reduced bioenergenesis associated with decreased glucose uptake in CP-r KB-CP20 and BEL7404-CP20 cells. CP-r KB-CP20 cells originally demonstrated 1152-fold more resistance to cisplatin than parental cells. Interestingly, the same cells showed 330-fold more resistance to cisplatin after incubation for 94 days without cisplatin selection pressure, which reduced SIRT1 expression (Shen, DW, Gottesman MM, unpublished data). SIRT1 overexpression allowed the parental CP-s KB-3-1 and BEL7404 cells to survive through limited nutrient incubation, and caused cellular resistance to cisplatin treatment in CP-s cells. This is consistent with the effect of reduced SIRT1 by siRNA, which sensitizes the CP-r cells to cisplatin treatment. Our present data shed light on how SIRT1, regulated by glucose homeostasis, may contribute to the development of tumor cell resistance to cisplatin, and provide insight that may help in the development of treatments to overcome drug resistance in cancer patients.