Statistical Analysis

Data are presented as mean ± SE. Two-tailed Student’s t test was used to analyze the difference between the means of the treatment and the control groups. Differences with a p value of less than 0.05 were considered statistically significant.

3. 結果 (Results)

3.1 The acquisition of Iressa resistance in NSCLC PC-9 cells

Lung cancer cell that have acquired resistance to Iressa may complicate future treatment. In order to define the effects of resveratrol on target therapy of lung cancer, we set up a EGFR tyrosine kinase inhibitor-resistant cell model. The Iressa-resistant cells were established and that were derived from the parental sensitive PC-9 cell line. These Iressa-resistant cells were selected by stepwise increasing the concentrations of Iressa. The concentrations of Iressa were started at 1 μM and achieved 10 μM. Several reports have been shown that the antitumor activity of EGFR tyrosine kinase inhibitor was associated with G1 phase arrest in cancer cells (88-90). In order to investigate the characters of Iress- resistant cells different from parental PC-9/WT cells, PC-9/WT and PC-9/IR cells were cultured in the absence (control) or presence of various concentration (1, 5, 10 μM) of Iressa for 48 hrs. After treatment with Iressa, cells were stained with propidium iodide (PI) and subjected to DNA profile analysis by flow cytometry. Treatment with Iressa induced G1 phase arrest of PC-9/WT cells, and increased the percentage of cells with sub-G1 DNA content, which means the cell death. However, we did not find alteration of cell cycle or increasing of sub-G1 cell population in PC-9/IR cells under the same experimental condition (Figure 1). To further confirm the EGFR tyrosine kinase inhibitor resistance, we performed MTS assay to analysis the inhibition of cell viability by Iressa on PC-9 lung cancer cells. Results showed that PC-9/IR cells were significantly resistant to Iressa than PC-9/WT cells in a concentration-dependent manner (Figure 2).

The IC50 of Iressa in PC-9/IR cells (>10 μM) was at least 10-fold higher then parental PC-9/WT cells (IC50, <1 μM). After that, we further evaluated the different expression of EGFR and downstream signaling proteins in PC-9/WT and PC-9/IR cells. Figure 3a showed that the expression of EGFR had no difference between PC-9/WT and PC-9/IR cells. Nevertheless, the protein expression of EGFR and downstream molecules were similar in parental PC-9 cells and Iressa-resistant cells (Figure 3B). In order to investigate the effects of resveratrol on cell viability in

lung cancer cells, PC-9/IR and PC-9/WT cells were treated with resveratrol for 48 hrs in indicated dosages (1.25, 2.5, 5, 10 μM). We found that resveratrol shows similar toxicity in both PC-9/WT and PC-9/IR cells (Figure 4). Treatment with resveratrol, even to 10 μM, did not cause significant reduction of cell viability in PC-9/WT and PC-9/IR cells. According to above data, we successfully established an Iressa-resistant lung cancer cell line, PC-9/IR, as the later study model.

3.2 Resveratrol-induced de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells

Recently, considerable attention has been also focused on resveratrol (3,5,4′-trans-trihydroxystilbene), a well-known natural polyphenol found in large amount in grapes (91), that has been reported to exert multiple biological activities (92) including anti-inflammatory (93), anti-oxidant (94), inhibition of platelet aggregation (95), antitumor (96), and induction of apoptosis (97). Remarkably, the cancer chemopreventive activity; Aggarwal et al. and Jang et al. of 2 represents an important add value and it seems to be strictly connected to the antitumor, and the proapoptotic effects (98-100). In 2010, Fukui and his research team reported that resveratrol sensitizes a number of cancer cell lines to several anti-cancer drugs, including paclitaxel (85).

EGFR tyrosine kinase inhibitor, such as Iressa, has provided dramatic clinical response for EGFR mutation lung cancer patients. However, its efficiency is limited by the development of drug resistance.

Above literatures lead us come out a hypothesis: whether resveratrol sensitize EGFR tyrosine kinase inhibitor and has the de-resistance effect on Iressa-resistance PC-9/IR cells. To this end, we selected the Iressa dose (2.5 μM) base on the finding from figure 2. Both of PC-9/WT and PC-9/IR cells were pretreated with vehicle or various dosages of resveratrol (1.25, 2.5, 5, 10 μM) for 4 hrs in serum-free medium. Thereafter, the cells were incubated with 2.5 μM of Iressa for another 48 hrs.

At the end of the incubation period, cell viability was analyzed by MTS assay. The results show that pre-treatment with non-toxic dosages of resveratrol increased Iressa-induced cell death in PC-9/IR cells (8.76% ± 1.56% cell death in vehicle + Iressa group; 48.71% ± 3.12% cell death in resveratrol

+ Iressa group, p<0.0001). The cell death of combined treatment with reveratrol and Iressa was increased at less 5-fold than Iressa treatment only in PC-9/IR cells (Figure 5).

These data suggested that resveratrol may sensitize and de-resistance of EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells, also provide a potential treatment strategy of EGFR tyrosine kinase inhibitor-resistant cancer patients.

3.3 The potential target molecular may involve in the resistance of lung cancer cells to Iressa

Resistance to anticancer drugs, not only chemotherapy drugs but also target therapeutic drugs is widely observed in lung cancer patients. This limitation of therapeutic potential provides a powerful stimulus for developing new therapeutic approaches. In our previous results, we showed that pre-treatment with resveratrol increased Iressa-induced cell death in Iressa-resistant lung cnacer cells. It suggested that resveratrol may sensitize EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells. Therefore, it is critical and timely to further evaluate the molecular mechanisms involved in resveratrol-induced de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells.

To this end, we used non-biased experiments to evaluate the potential targets may involves in resveratrol-mediated de-resistance of EGFR tyrosine kinase inhibitor in Iressa-resistant lung cancer cells. We performed two-dimensional gel electrophoresis analysis, oligo gene expression microarray analysis and microRNA microarray assay to analyze the differential expression of proteins, genes and microRNAs in PC-9/WT and PC-9/IR cells. We found some potential downstream targets may involve in the resistance of Iressa of lung cancer cells.

The different expression of proteins between PC-9/WT and PC-9/IR were shown in figure 6, table 2 and table 3. In figure 6, it showed the two-dimensional protein maps of the PC-9/WT cells and PC-9/IR cells. The proteins are separated by different isoelectric point and molecular weight. To identify the differential expression proteins from two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we punch out the target spots and then send to liquid chromatography and tandem mass spectrometry (LC–MS/MS) assay (cooperate with Genomics Research Center,

Academia Sinica). There were 15 candidate proteins significantly increase in PC-9/IR cells compared with PC-9/WT cells (Table 2). We also found 9 candidate proteins were dramatically decreased in PC-9/IR cells compared with PC-9/WT cells (Table 3). Through the prediction of these candidate proteins by IPI and SWISS-PROT databases, we selected several candidate proteins, such as CPT2 (Carnitine O-palmitoyltransferase 2), ACAA1 (Acetyl-CoA acetyltransferase), LDHA (L-lactate dehydrogenase A), HNRNPA2B1 (Heterogeneous nuclear ribonucleoproteins A2/B1) and APBEC3C (Probable DNA dC->dU-editing enzyme APOBEC-3C), to investigate whether these candidate proteins involve in Iressa resistance and resveratrol-mediated de-resistance of Iressa of lung cancer cells. PC-9/IR cells were infected with shLuc, shLDHA, shHNRNPA2B1, shACAA1, shAPOBOC3C and shCPT2 as described under “materials and methods” and were subjected to analysis the knockdown efficiency by real-time PCR quantification (Figure 7A). Data from western blot confirmed the decreased the expression of LDHA by shLDHA infection (Figure 7B). To further confirm the 2D prediction data, we examined the mRNA level and protein level of LDHA in PC-9/WT and PC-9/IR cells. As the results, the mRNA level of LDHA had no difference between PC-9/WT and PC-9/IR cells, but the expression of LDHA was higher in PC-9/IR cells then in PC-9/WT cells (Figure 8). To further evaluate the role of LDHA, HNRNPA2B1, ACAA1, APOBEC3C and CPT2, we used the stable cells of knockdown of 5 candidate proteins from figure 7a. These cells were treated with or without Iressa for 48 hrs. In the end of incubation period, cells were collected and analyzed the population of sub-G1 phase by flow cytometric assay. Figure 9 showed that knockdown of LDHA significantly increase the sub-G1 phase after treatment with Iressa in PC-9/IR cells. These data indicated that LDHA regulated the resistance of PC-9/IR to Iressa.

According to the oligo gene expression microarray analysis, there are some differential expressing genes between PC-9/WT and PC-9/IR cells. There were 14 candidate target genes significantly up-regulated in PC-9/IR cells (Ratio greater than 4 times, Table 4) and 3 candidate target genes were down-regulated in PC-9/IR cells (Ratio less than 0.25 times, Table 5). To further

confirm the expression of these candidate genes in PC-9/WT and PC-9/IR cells, we used RT-PCR to analysis the expression of these candidate genes in mRNA level. The mRNA level of IGFBP7 (Insulin-like growth factor binding protein7), SLC47A2 (Multidrug and toxin extrusion protein 2), FN1 (Fibronectin1) and GAS6 (Growth arrest-specific protein 6 Precursor) were significantly upregulated in PC-9/IR cells (Figure 11A). SPRR1B (Cornifin-B) was dramatically decreased in PC-9/IR cells (Figure 11B).

To further look for microRNAs potentially regulated the Iressa-resistance of lung cancer cells.

We analyzed the global microRNA expression in PC-9/WT cells and PC-9/IR cells using microRNA microarray. We only found 5 candidate microRNAs, miR-20a, miR-21, miR-23a, miR-200c and miR-574-5p, were upregulated (≧2 times) in PC-9/IR cells compared with PC-9/WT cells (Table 6). Through real-time PCR analysis, the differential expression of these up-regulated miRNAs in PC-9/IR cells was confirmed (Figure 12).

Summarized above results, we exposed the evident that resveratrol may sensitize Iressa and de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells those acquired drug-resistance.

Moreover, we found LDHA regulated the resistance of PC-9/IR to Iressa and might have other potential dowmstream molecules such as proteins, genes and microRNAs involve in Iressa-resistance.

4. 討論 (Discussion)

Our current study provided evidence for the first time that treatment with resveratrol sensitize EGFR tyrosine kinase, Iressa. We found pre-treatment resveratrol with non-toxic dosages, increased Iressa-induced cell death in drug-resistant lung cancer cells. Lung cancer is one of the most prevalent malignancies worldwide and remains the leading cause of cancer death (86). The EGFR tyrosine kinase inhibitors gefitinib and erlotinib are effective therapies for NSCLC patients.

However, the vast majority of gefitinib-responsive NSCLC ultimately progresses to a resistant state, and the emergence of this resistance severely limits the clinical efficiency of this drug. Therefore, it is necessary to develope a new therapeutic approach.

Resveratrol, a nature agent, has been shown to potentiate the apoptotic effects of cytokines, chemotherapeutic agents, and γ–radiation (101). Several studies found that cancer cells exposed to resveratrol were sensitized to Paclitaxel-induced apoptosis, and it was by way of modifying the expression of apoptotic regulator proteins (102). In present study, we therefore examined the combined-treatment effects of resveratrol and Iressa in Iressa-resistant cells. The present work has led to identify the molecular mechanisms of the resveratrol-induced de-resistance of EGFR tyrosine kinase inhibitor in lung cancer cells. To understand the signaling profiles will help us to know better about how resveratrol to sensitize EGFR tyrosine kinase inhibitor in resistant lung cancer and provide new anti-cancer therapeutic strategies for EGFR tyrosine kinase inhibitor-resistant lung cancer patients.

It is notably that the effects of resveratrol on chemosensitization are dynamic. Previous study revealed that, trans-resveratrol reduced cellular death in SH-SY5Y neuroblastoma cells exposed to Paclitaxel by inhibiting Paclitaxel-induced activation of caspase 7 and the degradation of poly (ADP-ribose) polymerase (103). The contrasting effects of resveratrol may be dose-dependent. It potentiates the effects of cytokines and chemotherapeutic agents at higher concentrations and inhibits their effects at lower concentrations. In the future, in animal model experiments, the

dosages of resveratrol should be more concerned.

There are many factors has been report that regulate the chemosensitization of resveratrol.

Resveratrol exerts its sensitizing effects by interruption with the cellular signaling pathways, induction of cell cycle arrest, and selective modification of apoptosis regulatory proteins (104). In our experiment, we discovered the novel molecular mechanisms involved in the regulating the chemosensitization of resveratrol to EGFR tyrosine kinase inhibitors. To this end, we performed non-bias experiments, such as two-dimensional gel electrophoresis analysis, oligo gene expression microarray analysis and microRNA microarray assay. Several targets which might regulate the resistance of lung cancer cells to Iressa were also be evaluated.

By two-dimensional gel electrophoresis analysis, the results show 5 candidate proteins may involved. Carnitine o-palmitoyltransferase 2 (CPT2), is a nuclear protein which is transported to the mitochondrial inner membrane. CPT2 together with carnitine palmitoyltransferase I oxidizes long-chain fatty acids in the mitochondria. Although the CPT2 involved in resveratrol-mediated de-resistance of EGFR-TKI is investigated yet, several papers were published that long-chain fatty acids suppress the developmentof major cancers (105-111). Acetyl-CoA acetyltransferase (ACAA1 or thiolase) is an enzyme which converts two units of acetyl-CoA to acetoacetyl CoA in the mevalonate pathway. Mevalonate metabolites play an essential role in transducing EGFR-mediated signaling. Mantha et al. reported that targeting the mevalonate pathway can inhibit EGFR function (112). Therefore, ACAA1 should be further investigating. L-lactate dehydrogenase A (LDHA) catalyzes the conversion of L-lactate and NAD⁺ to pyruvate and NADH in the final step of anaerobic glycolysis. According to the Warburg effect, cancer cells derive most of their energy from anaerobic glycolysis and that is contributed to the malignancy of cancer cells. Hence, the mechanism of drug-resistant was through LDHA proteins. Heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) belongs to hnRNPs. HnRNPs are RNA binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of

mRNA metabolism and transport. It has been reported, increased HNRNPA2B1 expression was found in lung cancer cells, indicated that HNRNPA2B1 are involved in cancer progression.

According to above literatures discussions, these proteins might regulate the resveratrol-mediated de-resistance of EGFR-TKI.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs (20-23 nucleotides) that negatively regulate the gene expressions at the post-transcriptional level by base pairing to the 3’

untranslated region (3’UTR) of target messenger RNAs. Evidence is emerging that particular microRNAs (miRNA) alterations are involved in the initiation and progression of human cancer.

More recently, accumulating evidence is revealing an important role of miRNAs in anticancer drug resistance and miRNA expression profiling can be correlated with the development of anticancer drug resistance.

It has been reported resveratrol could regulate the expression of miRNA. A study reported by Lukiw et al. has shown that resveratrol analog CAY10512 regulated the expression of miR-146a (113). They found that miR-146a was up-regulated and complement factor H (CFH), an important repressor of the inflammatory response in the brain, was down-regulated in the brain of Alzheimer disease. The sequence of miR-146a was highly complementary to the 3’UTR of CFH, suggesting that CFH is a target of miR-146a. Further experiments showed that transfection of human neural cells with pre-miRNA-146a promoter-luciferase reporter construct in stressed human neural cells showed significant up-regulation of luciferase activity and low level of CFH gene expression, consistent with the observations on the brain of Alzheimer disease. Importantly, treatment of stressed human neural cells with resveratrol analog CAY10512 or an anti-sense oligonucleotide to miRNA-146a could inhibit miR-146a and restore CFH expression levels (113). These data indicate that resveratrol could regulate the expression of specific miRNA and alter the signaling transduction, leading to the alterations in cell physiological behavior. Therefore, we found miR-21, miR-23a, miR-200c and miR574-5p were up-regulated in PC-9/IR, imply that those microRNAs have significantly potentials in regulation of Iressa-resistance.

In conclusion, we present the first evidence demonstrating that resveratrol, a polyphenols, may sensitize EGFR tyrosine kinase inhibitor, Iressa, in resistant lung cancer cells. It is necessary to understand the specific molecular mechanism, to improve the current anti-cancer treatment and to develop novel therapeutic strategies against cancer.

5. 參考文獻 (References)

1. Parkin, DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55: 74-108

2. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2009) The convergence of lung cancer rates between blacks and whites under the age of 40, United States. Cancer Epidemiol Biomarkers Prev 18: 3349-3352.

3. Spira A, Ettinger DS (2004) Multidisciplinary management of lung cancer. N Engl J Med 350:

379–392.

4. Schiller JH, Harrington D, Belani CP, Langer C, Sandler A, et al.; Eastern Cooperative Oncology Group (2002) Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 346: 92–98.

5. Ohe Y, Ohashi Y, Kubota K, Tamura T, Nakagawa K, et al. (2007) Randomized phase III study of cisplatin plus irinotecan versus carboplatin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for advanced non-small-cell lung cancer:

Four-Arm Cooperative Study in Japan. Ann Oncol 18: 317–323.

6. Nishio K, Nakamura T, Koh Y, Suzuki T, Fukumoto H, et al. (1999) Drug resistance in lung cancer. Curr Opin Oncol 11: 109-115.

7. Hynes NE, Lane HA (2005) ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 5: 341-354.

8. Mukohara T, Kudoh S, Yamauchi S, Kimura T, Yoshimura N, et al. (2003) Expression of epidermal growth factor receptor (EGFR) and downstream-activated peptides in surgically excised non-small-cell lung cancer (NSCLC). Lung Cancer 41: 123-130.

9. Herbst RS, Langer CJ (2002) Epidermal growth factor receptors as a target for cancer treatment: The emerging role of IMC-C225 in the treatment of lung and head and neck

cancer. Semin Oncol 29: 27–36.

10. Ethier SP (2002) Signal transduction pathways: The molecular basis for targeted therapies.

Semin Radiat Oncol 12: 3–10.

11. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, et al. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350: 2129–2139.

12. Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497–1500.

13. Mitsudomi T, Yatabe Y (2007) Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci 98: 1817-1824.

14. Giaccone G (2005) Epidermal growth factor receptor inhibitors in the treatment of non-small-cell lung cancer. J Clin Oncol 23: 3235–3242.

15. Inoue A, Suzuki T, Fukuhara T, Maemondo M, Kimura Y, et al. (2006) Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations. J Clin Oncol 24: 3340-3346.

16. Inukai M, Toyooka S, Ito S, Asano H, Ichihara S, et al. (2006) Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.

Cancer Res 66: 7854-7858.

17. Kobayashi S, BoggonTJ, DayaramT, Jänne PA, Kocher O, et al. (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352: 830-832.

18. Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, et al. (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR

kinase domain. PLoS Med 2: e73.

19. Herbst RS, Fukuoka M, Baselga J (2004) Gefitinib—a novel targeted approach to treating cancer. Nat Rev Cancer 4: 956–965.

20. Sharma SV, Bell DW, Settleman J, Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7: 169–181.

21. Negoro S, Nakagawa K, Fukuoka M, Kudoh S, Tamura T, et al. (2001) Final results of a phase I intermittent dose-escalation trial of ZD1839 (‘Iressa’) in Japanese patients with various solid tumours. Proc Am Soc Clin Oncol 20: 324.

22. Ranson M, Hammond LA, Ferry D, Kris M, Tullo A, et al. (2002) ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J Clin Oncol 20: 2217-2219.

23. Woodburn JR., Barker AJ, Gibson KH, Ashton SE, Wakeling AE, et al. (1997) ZD 1839, an epidermal growth factor tyrosine kinase inhibitor selected for clinical development. Proc

23. Woodburn JR., Barker AJ, Gibson KH, Ashton SE, Wakeling AE, et al. (1997) ZD 1839, an epidermal growth factor tyrosine kinase inhibitor selected for clinical development. Proc