Selective killing of K-ras–transformed pancreatic cancer cells by targeting NAD(P)H oxidase
© Wang et al.; licensee BioMed Central. 2015
Received: 17 August 2014
Accepted: 22 December 2014
Published: 8 April 2015
Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and plays a critical role in the pathogenesis of this malignancy. Increase of reactive oxygen species (ROS) has also been observed in a wide spectrum of cancers. This study aimed to investigate the mechanistic association between K-ras–induced transformation and increased ROS stress and its therapeutic implications in pancreatic cancer.
ROS level, NADPH oxidase (NOX) activity and expression, and cell invasion were examined in human pancreatic duct epithelial E6E7 cells transfected with K-ras G12V compared with parental E6E7 cells. The cytotoxic effect and antitumor effect of capsaicin, a NOX inhibitor, were also tested in vitro and in vivo.
K-ras transfection caused activation of the membrane-associated redox enzyme NOX and elevated ROS generation through the phosphatidylinositol 3′-kinase (PI3K) pathway. Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras–transformed cells compared with parental E6E7 cells. Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras–transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells. In vivo, capsaicin exhibited antitumor activity against pancreatic cancer and showed oxidative damage to the xenograft tumor cells.
K-ras oncogenic signaling causes increased ROS stress through NOX, and abnormal ROS stress can selectively kill tumor cells by using NOX inhibitors. Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras–transformed cells through a redox-mediated mechanism.
Oncogenic mutations of the KRAS gene are present in >90% of pancreatic ductal carcinoma , which is an aggressive and deadly cancer . Since pancreatic ductal carcinoma is unusually resistant to chemotherapy and radiation therapy and little progress has been achieved in the treatment of pancreatic cancer, surgical resection remains to be the only potentially curative therapy. The potential discoveries of pancreatic cancer therapeutics rely on advances in our understanding of the biology of the disease. Genetic lesions, including mutations of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein 53 (TP53), breast cancer 2 (BRCA2), and mothers against decapentaplegic homolog 4 (SMD4/DPC4), have been thought to contribute to the evolution of pancreatic adenocarcinoma . Activating KRAS mutations are found in more than 90% of pancreatic adenocarcinomas and are highly associated with disease progression due to the activation of several effector pathways that induce cell proliferation, survival, invasion, and metabolic alterations [3-5]. Given the almost ubiquitous occurrence of K-ras mutations and its critical role in the development of pancreatic cancer, the ideal therapeutic strategy would be the direct blocking of KRAS oncogenic signaling. However, an effective small-molecule inhibitor of KRAS has yet to be identified .
Whereas the major effector proteins, such as Raf kinase, phosphatidylinositol 3′-kinase (PI3K), and RalGDS, play vital roles in Ras transformation, accumulating evidence has shown that reactive oxygen species (ROS) may serve as a messenger of Ras in signaling transduction pathways and that moderate increases in ROS levels may promote cell proliferation and contribute to cancer development [7,8]. Therefore, ROS appear to be an important downstream effector of Ras transformation in cancer cells. The role of the membrane-associated NADPH oxidase (NOX) in non-mitochondrial formation of ROS has been observed in various studies [9-11]. The activation or up-regulation of NOX has also been shown to play an important role in maintaining the cancer phenotype through stimulating the production of ROS [12-14]. The previous findings prompted us to investigate whether K-ras oncogenic signaling increases ROS levels through the activation of NOX and whether modulators of NOX could provide a potential therapeutic opportunity for pancreatic cancer through a redox-mediated mechanism. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a natural compound, is a pungent ingredient found in a variety of red peppers and has been shown to inhibit cell surface NOX activity [15,16]. In the current study, we aimed to determine the mechanistic role of NOX in mediating ROS generation induced by K-ras oncogenic signaling. We compared ROS production as well as the expressions and activities of NOX in parental human pancreatic duct epithelial E6E7 cells and K-ras–transformed E6E7 cells, which have previously been shown to be highly tumorigenic . We also examined the effect of capsaicin on parental and K-ras–transformed E6E7 cells. Importantly, the role of NOX-derived ROS generation in capsaicin-induced cytotoxicity was tested in K-ras–transformed E6E7 cells in comparison with parental E6E7 cells.
Antibodies and reagents
The following antibodies were used for immunoblotting analysis using standard Western blotting procedures: superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), p22phox, and p-p40phox were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin, tublin, diphenyleneiodonium chloride (DPI), and capsaicin were purchased from Sigma-Aldrich, St. Louis, MS, USA.
The parental E6E7 cell line and K-ras–transformed cell line, which had been established by transfecting the immortalized human pancreatic duct epithelial E6E7 cell line with K-ras G12V, were kindly provided by Dr. Paul Chiao from The University of Texas, MD Anderson Cancer Center and were cultured as reported previously . Primary pancreatic cancer cell lines, including AsPC-1, Capan-1, and Panc-1, were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS).
Quantitative real-time Polymerase Chain Reaction (PCR) analysis
The sequences for the genes to be measured are as follows: 5′-GGAGTTTCAAGATGCGTGGAAACTA-3′ (sense) and 5′-GCCAGACTCAGAGTTGGAGATGCT-3′ (antisense) for NOX2, 5′-CAAGCCGTGACCAAGGACACCTG-3′ (sense) and 5′-CACACAGGACATCCACCGTGTC-3′ (antisense) for NOXA1. Real-time PCR analysis was performed by using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Otsu, Shiga, Japan) and Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA).
Cell growth was determined using MTT reagent in 96-well plates. After incubation, 20 μL MTT reagent was added to each well and incubated for an additional 4 hours and then the supernatant was removed. The cell pellets were dissolved in 200 μL DMSO. Absorbance was determined using a MultiSkan plate reader (Thermo, Helsinki, USA) at a wavelength of 570 nm.
Colony formation assay
Cells were seeded in six-well plates and cultured for about 2 weeks. Colonies were fixed with methanol for 10 minutes and stained with crystal violet solution (Beyotime, Jiangsu, China) for 30 minutes. All the experiment was repeated 3 times.
Cells were suspended in lysis buffer containing 20 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA,1 mmol/L EGTA, 100 mmol/L sucrose, and a cocktail of protease inhibitors. After homogenization, the samples were centrifuged at 800 g at 4°C for 5 minutes to pellet unbroken cells and nuclei. The supernatants were centrifuged at 100,000 g for 30 minutes to separate the membrane fraction (pellet) and the cytosolic fraction (supernatant). NOX activity was measured by lucigenin-derived chemiluminescence, with 100 μmol/L NADPH or NADH as substrate, 50 μmol/L lucigenin, and 25 μg of cell membrane proteins. Chemiluminescence was measured using a luminometer (Turner Designs, Sunnyvale, CA, USA) for 1 minute. The signal was normalized and expressed as arbitrary light units per microgram protein per minute.
The Rac activity assay was performed using the Rac-GEF (guanine-nucleotide exchange factors) Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, cells were washed in cold PBS, lysed in 1× Assay/Lysis Buffer, and centrifuged for 10 minutes at 14,000 g at 4°C. Aliquots from the supernatant were used for determining protein concentration. The supernatant was incubated with nucleotide-free Rac1 G15A agarose beads to pull down the active form of Rac-GEFs. The beads were washed 3 times with 1× Assay/Lysis Buffer, and the bound proteins were eluted. The active Rac proteins were detected by Western blotting using an anti-Rac-GEF antibody (Tiam1).
Invasion assays were performed with BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). Pre-coated filter Matrigel inserts were re-hydrated with 0.5 mL of PBS for 2 hours in humidified tissue culture incubator at 37°C in 5% CO2 atmosphere. After rehydration, PBS was removed. Then, 1 × 105 parental or K-ras–transformed E6E7 cells and Capan-1, AsPC-1, Panc-1 cells in 0.5 mL of supplement-free medium with or without 10 μmol/L capsaicin were seeded onto the upper part of each chamber insert, and the 24-well plates were filled with 0.5 mL of their culture medium. Following incubation for 16 hours, non-invaded cells on the upper surface of the insert were wiped off with a cotton swab, and the cells that had migrated onto the lower surface of the filter, were fixed and stained with the Hema 3 Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA) containing a fixative and 2 stain solutions. The inserts were air dried and photographed. Invasiveness was determined by counting cells in 3 microscopic fields (×100) per well, and the extent of invasion was expressed as an average number of cells per microscopic field.
Measurement of ROS production and ATP generation
Cells were stained with 100 ng/mL hydroethidine (HET) (Invitrogen, Carlsbad, CA, USA) and 5 μmol/L DCF-DA (Invitrogen, Carlsbad, CA, USA) for 60 minutes before the measurement of superoxide and hydrogen peroxide using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cellular ATP generation was measured using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Wisconsin, USA) according to manufacturer’s recommendations.
Four-week-old, BALB/c male nude mice were purchased from Medical Experimental Animal Center of Guangdong Province, China. A total of 2 × 106 AsPC-1 cells were inoculated into the right flanks of the mice by subcutaneous injection. When the volume of tumors reached 100 mm3, the mice were randomly divided into 2 groups of 10 mice each. The treatment group received 15 mg/kg capsaicin in 0.9% sodium chloride solution (intraperitoneal injection, 3 times per week). The control group received equal volume of 0.9% sodium chloride solution by intraperitoneal injection. Five weeks after inoculation, all mice were euthanized and the tumor weights were measured. Animal experiments were approved by Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center and performed under the guidelines of the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996).
Immunohistochemistry and TUNEL assay
Representative tumor tissues were sectioned and embedded in paraffin. The slides were then incubated with the primary antibody (mouse anti–8-oxoguanine monoclonal antibody, Abcam, Cambridge, UK) at 1:200 dilution overnight in a humidified chamber at 4°C. The slides were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Envision Detection Kit, Dako, Glostrup, Denmark) at 37°C for 30 minutes. Finally, the samples were stained with 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin and eosin (HE). Tumor cell death induced by capsaicin was detected by TUNEL assay with the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions.
Statistical significant differences were evaluated by using Student’s t test (Prism GraphPad, San Diego, CA, USA). The Kolmogorov-Smirnov test (Cell Quest Pro software, Becton-Dickinson, San Jose, CA, USA) was used to evaluate the significant difference between control and treatment groups in flow cytometry analysis. A P value of <0.05 was considered statistically significant.
Oncogenic transformation induced by K-ras increased ROS generation
As NOX has been reported to be a multi-subunit redox enzyme that generates non-mitochondrial source of ROS [10,18], in the current study, we examined the effect of K-ras transformation on NOX expression and enzyme activity. The mRNA levels of 2 members of the NOX family, NOX2 and NOXA1, were up-regulated by more than 3-fold in K-ras–transformed cells (Figure 1D). The activation of the NOX complex requires proper assembly of the plasma membrane-binding and cytosolic protein components, and the activation is initiated by phosphorylation of the cytosolic complex [19,20]. Western blotting analysis showed that p22phox, the major membrane-binding component, was markedly increased in the transformed cells. Importantly, the level of p40phox, the phosphorylated form of the cytosolic subunit, was also up-regulated in the transformed cells (Figure 1E). The K-ras–transformed cells also exhibited a more than 2-fold increase in the enzyme activity of NOX (Figure 1F) compared with parental E6E7 cells, indicating that NOX was indeed activated in the transformed cells.
Capsaicin preferentially induced ROS production and selectively inhibited NOX activity in K-ras–transformed cells
To determine the biochemical basis for the selective effect of capsaicin on the context of ROS generation in K-ras–transformed cells, NOX activity was assessed before and after capsaicin treatment. NOX is a multi-subunit enzyme, and the regulatory components, p40phox, p47phox, and p67phox, as well as the small GTPase, Rac, are localized in the cytosol during the resting state . Upon activation, GDP is exchanged for GTP, and Rac dissociates from the cytosolic complex, leading to the activation of NOX activity . Western blotting analysis showed that the level of functionally active form of Rac was up-regulated in K-ras–transformed cells. Importantly, the activation of Rac was significantly inhibited after 12 hours of treatment with10 μmol/L capsaicin (Figure 2E). In accordance with the inhibition of active Rac by capsaicin, NOX activity in K-ras–transformed cells was also suppressed by approximately 50% when they were treated with 10 μmol/L capsaicin for 12 hours (Figure 2F).
Capsaicin induced selective cytotoxicity in K-ras–transformed cells
Because pancreatic cancer is also known to be an aggressive cancer with enhanced invasiveness, the effects of capsaicin on cell migration and invasion were also tested in parental and K-ras–transformed E6E7 cells. The invasion measurement was performed with BD chamber inserts coated with a layer of Matrigel matrix. The invasive ability was measured by counting the number of cells that digested the Matrigel and passed through the filter membrane. Consistently, the number of K-ras–transformed cells with invasive ability was larger than that of parentalE6E7 cells (Figure 3E). After 24 hours of treatment with 10 μmol/L capsaicin, the invasive ability of K-ras–transformed E6E7 cells was significantly inhibited (Figure 3E). The treatment of capsaicin for 24 h inhibited the invasion of K-ras–transformed cells by more than 70% (P < 0.01) (Figure 3F). Taken together, capsaicin not only showed selective cytotoxicity but also inhibited invasion in malignant cells, either K-ras–transformed pancreatic cancer cells or primary pancreatic cancer cells, whereas both effects were not shown in parental E6E7 cells.
Capsaicin effectively suppressed the proliferation of pancreatic cancer cells and exhibited antitumor activity in vivo
The association between K-ras mutation and pancreatic cancer has been known for decades. An increase in oxidative stress in cancer cells and its association with cancer progression has also long been recognized. However, the mechanistic link between K-ras transformation and intrinsic oxidative stress in pancreatic cancer and its therapeutic implications remain to be investigated.
Using isogenic cell lines, we showed here that compared with the parental human pancreatic duct epithelial E6E7 cells, K-ras–transformed pancreatic cancer cells exhibited a substantial increase in basal ROS levels, including both O2 − and H2O2, indicating that the primary elevated generation of O2 − may lead to an increase in H2O2 due to intracellular conversion. Accordingly, we found a significant up-regulation of both SOD1 and SOD2 in the K-ras–transformed cells compared with parental E6E7 cells.
Therapeutic selectivity, or the preferential killing of cancer cells without significant toxicity to normal cells, is an important consideration in cancer chemotherapy. In the current study, the constitutive activation of NOX was observed in K-ras–transformed pancreatic cancer cells. The deregulation and overexpression of the NOX family members have been reported in a variety of malignancies, and certain subunits seem to be associated with K-ras transformation [4,13,30]. Interestingly, we recently reported an up-regulation of p22phox in clinical pancreatic cancer specimens , and Hiraga et al.  reported that NOX4-derived ROS signaling contributes to TGF-β–induced epithelial-mesenchymal transition (EMT) in pancreatic cancer cells. Therefore, NAD(P)H oxidase may be an attractive target for preferentially killing malignant cells and is likely to have broad therapeutic implications. Capsaicin, a natural compound found in peppers, has been found to interact with NOX and induce ROS generation . Our study revealed that capsaicin caused selective cytotoxicity to K-ras–transformed cancer cells but minimum effect on parental E6E7 cells. Capsaicin was also able to inhibit the proliferation of primary pancreatic cancer AsPC-1 cells. In addition, capsaicin was able to inhibit invasion, the deadly feature of pancreatic cancer, in both K-ras–transformed pancreatic cancer cells and primary pancreatic cancer cell lines, Capan-1, Panc-1, and AsPC-1. Importantly, capsaicin induced a tremendous accumulation of ROS in K-ras–transformed cells compared with parental E6E7 cells. The in vitro assay of NOX activity showed that capsaicin preferentially inhibited NOX activity through the suppression of Rac, a NOX component, in K-ras–transformed cells, whereas only having minimal effects on parental E6E7 cells. DPI, another NOX inhibitor, also exhibited a selective depletion of ATP in K-ras–transformed pancreatic cancer cells as well as primary pancreatic cancer cells. Furthermore, our animal study showed that capsaicin has therapeutic activity in mice bearing pancreatic cancer xenografts. Examination of the tumor sections demonstrated the oxidative damage induced by capsaicin and suggesting the role of ROS accumulation in capsaicin-induced cell death.
Compared with normal cells, cancer cells are under sustained oxidative stress due to the presence of constant oncogenic signals and hence are highly dependent on antioxidants to counterbalance ROS stress . Therefore, further oxidative insults, such as the exposure to ROS-generating agents, could exhaust the cellular antioxidant capacity and cause severe accumulation of ROS, leading to cell death. In contrast, it is less likely to induce such severe ROS stress in normal cells, due to their low basal ROS levels. This biochemical difference between normal and cancer cells may constitute a basis for modulating cellular ROS as a strategy to selectively kill cancer cells. Our study demonstrated the significant increase in ROS in K-ras–transformed cells compared with parental E6E7 cells. Importantly, capsaicin induced further ROS accumulation in K-ras–transformed cells, but had little effects on parental E6E7 cells. Consistently, capsaicin significantly inhibited the growth and invasion of K-ras–transformed cells, whereas the identical concentration did not show any toxicity on parental E6E7 cells.
In conclusion, our results demonstrated the up-regulation and activation of NOX, the ROS-generating enzyme, by K-ras transfection. Our study suggests that the intrinsic oxidative stress associated with K-ras–induced oncogenic transformation provides a basis for developing strategies to specifically target pancreatic cancer cells through a redox-mediated mechanism. Considering the frequent mutation of K-ras in pancreatic cancer and its resistance to many anticancer agents, targeting NOX may have significant clinical implications and warrant further investigation.
This work was supported by research grants from the National Natural Science Foundation of China (No. 81101520) and the National Basic Research Program of China (973 program) (No. 2013CB910304 and 2012CB967004).
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