9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

Transduction efficacy of Tat-Cu,Zn-superoxide dismutase is enhanced by copper ion recovery of the fusion protein

We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. To enhance the therapeutic potential of Tat-SOD for the treatment of various disorders that are related to this antioxidant enzyme, the transduction efficacy of Tat-SOD should be heightened. Therefore, we investigated whether copper ion recovery of the fusion protein could enhance the transduction potential of Tat-SOD in cultured HeLa cells. The results showed that the transduction potential of Tat-SOD was markedly enhanced by copper ions, and moderately increased by zinc ions. Compared with Tat-SOD, the Tat-SOD that recovered the copper ion (CR-Tat-SOD) achieved a significant increase in intracellular concentration and enzymatic activity. Therefore, CR-Tat-SOD was transduced into HeLa cells in a rapid saturation manner, but Tat-SOD was shown in a time-dependent manner. With the higher transduction efficacy of CR-Tat-SOD than that of Tat-SOD, the transduced CR-Tat-SOD significantly increased the viability of HeLa cells that were pretreated with paraquat, an intracellular superoxide anion generator. Although the mechanism of the enhanced transduction of Tat-SOD by copper ions is still unanswered, these results indicate that copper ions facilitate the transduction of SOD. These then significantly increase the biological effectiveness of this antioxidant enzyme.     

In vivo protein transduction: biologically active intact pep-1-superoxide dismutase fusion protein efficiently protects against ischemic insult

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD) is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that denatured Tat-SOD fusion protein is transduced into cells and skin tissue. Moreover, PEP-1 peptide, which has 21 amino acid residues, is a known carrier peptide that delivers full-length native proteins in vitro and in vivo. In the present study, we investigated the protective effects of PEP-1-SOD fusion protein after ischemic insult. A human SOD gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD fusion protein. The expressed and purified fusion proteins were efficiently transduced both in vitro and in vivo with a native protein structure. Immunohistochemical analysis revealed that PEP-1-SOD injected intraperitoneally (i.p.) into mice can have access into brain neurons. When i.p.-injected into gerbils, PEP-1-SOD fusion proteins prevented neuronal cell death in the hippocampus caused by transient forebrain ischemia. These results suggest that the biologically active intact forms of PEP-1-SOD provide a more efficient strategy for therapeutic delivery in various human diseases related to this antioxidant enzyme or to ROS, including stroke.     

Enhanced transduction of Cu,Zn-superoxide dismutase with HIV-1 Tat protein transduction domains at both termini

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.     

Suppression of arsenic trioxide-induced apoptosis in HeLa cells by N-acetylcysteine

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).     

Systemic administration of PEP-1-SOD1 fusion protein improves functional recovery by inhibition of neuronal cell death after spinal cord injury

Spinal cord injury (SCI) produces excessive levels of reactive oxygen species (ROS) that induce apoptosis of neurons. Cu,Zn-superoxide dismutase (SOD1) is a key antioxidant enzyme that detoxifies intracellular ROS, thereby protecting cells from oxidative damage. PEP-1 is a peptide carrier capable of delivering full-length native peptides or proteins into cells. In the study described here, we fused a human SOD1 gene with PEP-1 in a bacterial expression vector to produce a genetic in-frame PEP-1-SOD1 fusion protein; we then investigated the neuroprotective effect of the fusion protein after SCI. The expressed and purified PEP-1-SOD1 was efficiently delivered into cultured cells and spinal cords in vivo, and the delivered fusion protein was biologically active. Systemic administration of PEP-1-SOD1 significantly decreased levels of ROS and protein carbonylation and nitration in spinal motor neurons after injury. PEP-1-SOD1 treatment also significantly inhibited mitochondrial cytochrome c release and activation of caspase-9 and caspase-3 in spinal cords after injury. Furthermore, PEP-1-SOD1 treatment significantly reduced ROS-induced apoptosis of motor neurons and improved functional recovery after SCI. These results suggest that PEP-1-SOD1 may provide a novel strategy for the therapeutic delivery of antioxidant enzymes that protect neurons from ROS after SCI.     

Protein transduction of an antioxidant enzyme: subcellular localization of superoxide dismutase fusion protein in cells

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.     

Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells

Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B6 to pyridoxal-5'-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-PK fusion protein. The expressed and purified Tat-PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat-PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat-PK to the PC12 cells. Those results suggest that the transduction of Tat-PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.     

Transduced Tat-SOD fusion protein protects against ischemic brain injury

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.     

Ginsenosides enhance the transduction of tat-superoxide dismutase into mammalian cells and skin

We previously reported that Tat-Cu,Zn-superoxide dismutase (Tat-SOD), a major antioxidant enzyme, can be directly transduced into mammalian cells and skin [Kwon et al. (2000); Park et al. (2002)]. To enhance the therapeutic potential of Tat-SOD in the treatment of various disorders, we screened a number of natural products for their ability to increase transduction efficiency. Ginsenosides were effective with cultured HeLa cells and enhanced the penetration of Tat-SOD into both the epidermis and the dermis of the subcutaneous layer when sprayed on mice skin. Although their mechanism of action is not fully understood we believe that ginsenosides may be useful cofactors with this antioxidant enzyme in anti-aging cosmetics or as a therapeutic protein in disorders related to reactive-oxygen species.     

Transduced human PEP-1–catalase fusion protein attenuates ischemic neuronal damage

Antioxidant enzymes are considered to have beneficial effects against various diseases mediated by reactive oxygen species (ROS). Ischemia is characterized by both oxidative stress and changes in the antioxidant defense system. Catalase (CAT) and superoxide dismutase (SOD) are major antioxidant enzymes by which cells counteract the deleterious effects of ROS. To investigate the protective effects of CAT, we constructed PEP-1–CAT cell-permeative expression vectors. When PEP-1–CAT fusion proteins were added to the culture medium of neuronal cells, they rapidly entered the cells and protected them against oxidative stress-induced neuronal cell death. Immunohistochemical analysis revealed that PEP-1–CAT prevented neuronal cell death in the hippocampus induced by transient forebrain ischemia. Moreover, we showed that the protective effect of PEP-1–CAT was observed in neuronal cells treated with PEP-1–SOD. Therefore, we suggest that transduced PEP-1–CAT and PEP-1–SOD fusion proteins could be useful as therapeutic agents for various human diseases related to oxidative stress, including stroke.     

HIV-1 Tat-mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo

Reactive oxygen species (ROS) are considered an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, we investigated the HIV-1 Tat protein transduction domain-mediated transduction of Cu,Zn-superoxide dismutase (SOD), which supplies SOD activity exogenously in pancreatic beta cells under oxidative stress. Tat-SOD fusion protein was successfully delivered into insulin-producing RINm5F cells and rat islet cells. The intracellular dismutation activities of SOD were found to increase in line with the amount of protein delivered into the cells. ROS, nitric oxide-induced cell death, lipid peroxidation, and the DNA fragmentation of insulin-producing cells were found to be significantly reduced when the cells were pretreated with Tat-SOD. Next, we examined the in vivo transduction of Tat-SOD into streptozotocin-induced diabetic mice. A single intraperitoneal injection of Tat-SOD resulted in the delivery of this biologically active enzyme to the pancreas. Moreover, increased radical scavenging activity in the pancreas was induced by multiple injections of Tat-SOD, and this enhanced the tolerance of pancreatic beta cells to oxidative stress. These results suggest that the transduction of Tat-SOD offers a new strategy for protecting pancreatic beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes.     

A fused selenium-containing protein with both GPx and SOD activities

As a safeguard against oxidative stress, the balance between the main antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was believed to be more important than any single one, for example, dual-functional SOD/CAT enzyme has been proved to have better antioxidant ability than either single enzyme. By combining traditional fusion protein technology with amino acid auxotrophic expression system, we generated a bifunctional enzyme with both GPx and SOD activities. It displayed better antioxidant ability than GPx or SOD. Such dual-functional enzymes could facilitate further studies of the cooperation of GPx and SOD and generation of better therapeutic agents.     

Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species

Cancer cells have been found to express immunoglobulin G (IgG), but the exact functions and underlying mechanisms of cancer-derived IgG remain elusive. In this study, we first confirmed that downregulation of IgG restrained the growth and proliferation of cancer cells in vitro and in vivo. To elucidate its mechanism, we carried out a co-immunoprecipitation assay in HeLa cells and identified 27 potential IgG-interacting proteins. Among them, receptor of activated protein kinase C 1 (RACK1), ras-related nuclear protein (RAN) and peroxiredoxin 1 (PRDX1) are closely related to cell growth and oxidative stress, which prompted us to investigate the mechanism of action of IgG in the above phenomena. Upon confirmation of the interactions between IgG and the three proteins, further experiments revealed that downregulation of cancer-derived IgG lowered levels of intracellular reactive oxygen species (ROS) by enhancing cellular total antioxidant capacity. In addition, a few ROS scavengers, including catalase (CAT), dimethylsulfoxide (DMSO), n-acetylcysteine (NAC) and superoxide dismutase (SOD), further inhibited the growth of IgG-deficient cancer cells through suppressing mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK) signaling pathway induced by a low level of intracellular ROS, whereas exogenous hydrogen peroxide (H₂O₂) at low concentration promoted their survival via increasing intracellular ROS levels. Similar results were obtained in an animal model and human tissues. Taken together, our results demonstrate that cancer-derived IgG can enhance the growth and proliferation of cancer cells via inducing the production of ROS at low level. These findings provide new clues for understanding tumor proliferation and designing cancer therapy.     

A comparative proteomic analysis for capsaicin-induced apoptosis between human hepatocarcinoma (HepG2) and human neuroblastoma (SK-N-SH) cells

The endogenous ROS levels were increased during HepG2 apoptosis, whereas they were decreased during SK-N-SH apoptosis in response to capsaicin treatments. We used 2-DE-based proteomics to analyze the altered protein levels in both cells, with special attention on oxidative stress proteins before and after capsaicin treatments. The 2-DE analysis demonstrated that 23 proteins were increased and 26 proteins were decreased significantly (fold change>1.4) in capsaicin-treated apoptotic HepG2 and SK-N-SH cells, respectively. The distinct effect of capsaicin-induced apoptosis on the expression pattern of HepG2 proteins includes the downregulation of some antioxidant enzymes including aldose reductase (AR), catalase, enolase 1, peroxiredoxin 1, but upregulation of peroxiredoxin 6, cytochrome c oxidase, and SOD2. In contrast, most antioxidant enzymes were increased in SK-N-SH cells in response to capsaicin, where catalase might play a pivotal role in maintenance of low ROS levels in the course of apoptosis. The global gene expression for oxidative stress and antioxidant defense genes revealed that 84 gene expressions were not significantly different in HepG2 cells between control and capsaicin-treated cells. In contrast, a number of oxidative genes were downregulated in SK-N-SH cells, supporting the evidence of low ROS environment in apoptotic SK-N-SH cells after capsaicin treatment. It was concluded that the different relationship between endogenous ROS levels and apoptosis of two cancer cells presumably resulted from complicated expression patterns of many oxidative stress and antioxidant genes, rather than the individual role of some classical antioxidant enzymes such as SOD and catalase.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.     

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.     

Trolox enhances curcumin's cytotoxicity through induction of oxidative stress

Curcumin, a natural polyphenol in the spice turmeric, has been found to exhibit anticancer activity. Although curcumin is generally considered an antioxidant, it is also able to elicit apoptosis through the generation of ROS, thereby functioning as a pro-oxidant in cancer cells. The present study investigated the effects of antioxidant pretreatment on curcumin-induced cytotoxicity in the human cancer cell lines A2780, MCF-7, and MDA-MB-231. Cytotoxicity was enhanced by trolox, vitamin C or vitamin E; trolox, a water soluble vitamin E derivative, was the most potent. The combination of curcumin (10 μM) and trolox (10-50 μM) induced apoptosis of cancer cells as evidenced by PARP cleavage and caspase-3 activation. Furthermore, expression of the pro-apoptotic protein Bad was up-regulated and expression of the anti-apoptotic proteins Bcl-2 and Bcl-xl was down-regulated in cells that had been treated with trolox plus curcumin. ROS generation was detected in curcumin-treated cells and was significantly enhanced when cells were treated with trolox plus curcumin. Exogenous catalase or SOD1 did not alter cytotoxicity, while over-expression of either catalase or SOD1 did, pointing to the importance of intracellular hydrogen peroxide generation in cell killing. In conclusion, we demonstrated for the first time that antioxidants such as trolox can potentiate cancer cell killing by curcumin, a finding which may help in the development of novel drug combination therapies.