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Regulatory Mechanisms of Annexin-Induced Chemotherapy Resistance in Cisplatin Resistant Lung Adenocarcinoma

Regulatory Mechanisms of Annexin-Induced Chemotherapy Resistance in Cisplatin Resistant Lung... Adenocarcinoma of lung has high incidence and a poor prognosis, woith chemotherapy as the main therapeutic tool, most commonly with cisplatin. However, chemotherapy resistance develops in the majority of patients during clinic treatment. Mechanisms of resistance are complex and still unclear. Although annexin play important roles in various tumor resistance mechanisms, their actions in cisplatin-resistant lung adenocarcinoma remain unclear. Preliminary studies by our group found that in cisplatin-resistant lung cancer A549 cells and lung adenocarcinoma tissues, both mRNA and protein expression of annexins A1, A2 and A3 is increased. Using a library of annexin A1, A2 and A3 targeting combined molecules already established by ourselves we found that specific targeting decreased cisplatin-resistance. Taken together, the underlined effects of annexins A1, A2 and A3 on drug resistance and suggest molecular mechanisms in cisplatin-resistant A549 cells both in vivo and in vitro. Furthermore, the study points to improved research on occurrence and development of lung adenocarcinoma, with provision of effective targets and programmes for lung adenocarcinoma therapy in the clinic. Keywords: Annexin - lung adenocarcinoma - chemotherapy - effective targets Asian Pac J Cancer Prev, 15 (7), 3191-3194 studies indicate that Annexins (A1, A2, and A3) play Introduction significant roles in the growth of various tumors and the Lung cancer, with its rapidly growing morbidity, is production of drug resistance, which mark good target one of the common malignancies that ranks among the spots for tumor treatment (Mussunoor and Murray, 2008; top in the incidence of cancer among adults, and has Protzel et al., 2010). become the most common cause of human death from However, reports on the functions and mechanisms of tumors (Sun et al., 2014). Adenocarcinoma accounts action of Annexins (A1, A2, and A3) on adenocarcinoma for 40% of lung cancer incidence (Langer et al., 2010). and cisplatin-resistance have not been published. This Adenocarcinoma cannot be treated adequately through experiment aims to study the mechanisms of Annexins simple operations. Therefore, chemotherapy remains (A1, A2, and A3) in inhibiting cisplatin induced the mainstay treatments for lung cancer (Wang et al., apoptosis of adenocarcinoma cells, and primarily screen 2013; Zhou et al., 2014). Cisplatin, as the first line drug, and construct compound molecule libraries that can be is extensively applied for the clinical treatment of non- combined with Annexins (A1, A2, and A3)through crystal small-cell lung cancer (Fan and Jiang, 2008). However, diffraction and molecular space simulation. the drug resistance produced by tumor cells marks not only the most universal and difficult problem that influences Materials and Methods the curative effect of chemotherapy, but also the main reason that leads to chemotherapy failure. However, Cell culture, DDP resistant A549 stable cell line and no therapeutic schedule or drug effectively inhibits the primary cell culture from lung adenocarcinoma patients tolerance of adenocarcinoma and extend the life of patients A549 cells were maintained in RPMI supplemented have been developed so far (Zhang and Hu, 2006; Stewart with 10% fetal bovine serum, 2 mmol/l of glutamine, 100 et al., 2007; Stewart, 2010). Therefore, conducting in- units/ml of penicillin and 100 mg/ml of streptomycin in depth research on the new action mechanism of cisplatin a 5% CO atmosphere at 37°C. A549 cells were treated on adenocarcinoma and discovering key molecules that with DDP (1 ug/ml) for 48h, once a month, 6 times. A549/ play significant roles may lead to more effective candidate DDP cells resistant index for DDP is 18, meanwhile, with targets and treatment plans for adenocarcinoma. Many cross resistance to Carboplatin. 1 2 & Department of Pharmacy, Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Equal contributors *For correspondence: [email protected], [email protected] Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 3191 Chao Wang et al RT-PCR therapy for cisplatin resistance. Therefore, screening Expression of AnnexinA1, A2, A3 were analyzed by through Crystallography and Molecular space simulation, semiquantitative RT-PCR analysis. After the total RNA we preliminarily constructed libraries of molecular was prepared using a TRIZOL reagent (Life Technologies, compounds which could bind with Annexin A1, A2 and Inc., 15596-026), the oligo (dT)-primed cDNA was A3 respectively. Three compounds: compound1, 2 and 3 synthesized using a RT-PCR kit (Agilent Technologies, which binding with Annexin A1, A2 and A3 respectively 600182). The primers used in this study were summarized were selected and the structures are shown here (Figure in Table 1. 3). Annexin A1, A2 and A3 are all small molecule compounds, both A1 and A3 contain a benzo seven- Immunoblot analysis membered-ring. A2 contains a benzo five-membered- After treatment with different conditions as described ring, A1 and A3 contain an amide bond with α-amino. in the figure legends, cells were lysed in M2 buffer (20 Treatment of A549/DDP cells with compound 1, 2 and mM Tris at pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM 3, indeed, A549/DDP cells viability were dramatically EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 decreased (Figure 4). The result confirmed effectiveness of mM β-glycerol phosphate, 1 mM sodium vanadate, 1 mg/ ml leupeptin). Fifty micrograms of the cell lysates were subjected to SDS-polyacrylamide gel and blotted onto PVDF membrane (Millipore, PVH00010). After blocking with 5% skim milk in TBS/T, the membrane was probed with the relevant antibody and visualized by enhanced chemiluminescence (ECL, RPN2106), according to the manufacturer’s instruction (Amersham). Immunofluorescence analysis HeLa cells were grown on glass coverslips and then infected with Ad-GFP-LC3. After 24h, the cells were treated as indicated in the figure legend and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with PBS containing 0.2% Triton X-100 and 0.1 M glycine for 15 min, blocked with 10%BSA in PBS for 1 h and then incubated with rat anti-human Figureure 1. High Expression of Annexin A1, A2 and Statistical analysis A3 in A549/DDP Cells. mRNA expression of Annexin A1,A2 Data are expressed as the mean±SD from at least three and A3 in A549 and A549/DDP cells (left). Relative intensity of band were analyzed (right); Protein expression of Annexin A1, separate experiments performed triplicate. The differences A2, A3 in A549 and A549/DDP cells (left). Relative intensity between groups were analyzed using Student’s t-test. of band were analyzed (right) p<0.05 is considered statistically significant. Statistical analyses were performed using SPSS software ver. 13.0 (SPSS, Inc.). Results Firstly, in order to identify involvement of Annexins in cisplatin resistance, we constructed DDP resistant A549 lung adenocarcinoma stable cell line (A549/DDP), and compared expression of Annexin A1, A2 and A3 to A549 and A549/DDP in both mRNA and protein levels. Annexin A1, A2 and A3 highly expressed in A549/DDP cells (Figure 1 A, B), which suggested expression of Annexin A1, A2 and A3 closely related to cisplatin resistance. To further reveal the critical role of Annexin A1, A2 and A3 in cisplatin resistance in vivo, we observed the expression of Annexin A1, A2 and A3 in tumor tissues of lung adenocarcinoma patients. In consistent with in vitro data, expression of Annexin A1, A2 and A3 were up-regulated Figureure 2. High Expression of Annexin A1, A2 and in cisplatin resistant patients’ tissues both in mRNA and A3 in Cisplatin Resistant Cells From Tumors of Lung protein levels (Figure 2). Taken together, Annexin A1, A2 Adenocarcinoma Patients. mRNA protein expression and A3 overexpressed both in vitro and in vivo, suggested of Annexin A1, A2 and A3 in cisplatin resistant cells from they are most likely to be key regulator of cisplatin tumors of lung adenocarcinoma patients (lane1,3,5) and non- resistance, thus pharmacological approaches targeting cisplatin resistant cells from tumors of another part of lung to Annexin A1, A2 and A3 are considered as effective adenocarcinoma patients (lane2,4,6). 3192 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.7.3191 Annexin-Induced Chemotherapy Resistance in Cisplatin Resistant Lung Adenocarcinoma ductal adenocarcinoma, and is related to tumor invasion, metastasis, and prognosis. Moreover, previous studies reported that high A2 expression is related to the degree of malignancy, as well as invasion and metastasis (Emoto et al., 2001; Esposito et al., 2006; Sharma et al., 2006; Yee et al., 2007; Duncan et al, 2008). A2 is selectively Figureure 3. Hypothetic Molecular Structures of expressed in lung adenocarcinoma cell lines. Furthermore, Annexins Binding Compounds. Screening and construction of Annexin A1, A2 and A3 binding compounds libraries A2 promotes the invasion and metastasis of lung cancer respectively through Crystallography and Molecular space cells. However, no statement has been made regarding its simulation (ACCELRYS DISCOVERY STUDIO V1.6) relationship with cisplatin-resistance in adenocarcinoma (Gillette et al., 2004; Huang et al., 2008). Meanwhile, recent research results indicated that A3 expression in the tissues of patients with cisplatin-resistant prostate cancer and ovarian cancer has increased significantly. In addition, the cycles of patients without tumors in the A3 high expression group are clearly shortened (Gillette et al., 2004; Huang et al., 2008) Related studies also prove that A3 produces drug resistance by reducing the platinum content, the platinum-DNA combining quantity within cells, and the p53 level in ovarian cancer cells (Yan et al., Figureure 4. Effect of Annexins Binding Compounds 2010). The tumorigenesis of cells with high A3 expression on A549/DDP Cells. A549/DDP cells were treated with is significantly increased after cisplatin treatment, as compound1 (10 ug/ml), compound2 (10 ug/ml), compound3 demonstrated through an animal experiment. In addition, (10 ug/ml) vevfrom libraries respectively, cell viability was the resistance of cells with high A3 expression is clearly determined with MTT enhanced against platinum-based medicines; however, it Annexin A1, A2 and A3 targeting compounds in inducing does not induce cross-resistance with TAX and EPI (Yan cisplatin resistant A549 cell death. Furthermore, our study et al., 2010). provided powerful possibility of the concept of targeting Platinum resistance mechanisms, which are still the Annexin A1, A2 and A3 for the treatment of cisplatin unclear, mainly involve the following aspects: 1. Reduced resistant lung adenocarcinoma patients drug accumulation within tumor cells, including reducing cellular drug intake and increasing active efflux; 2. Functions of tumor cells to withstand the transformation Discussion of cancer medicines, as well as enhanced detoxification; The annexin family is super family of calphobindin, that 3. Strengthened DNA repair by tumor cells; 4. In the present in the intracellularly among major eucaryons, and induction of apoptosis by platinum-based medicines, the play crucial physiologic functions, such as participating expression of regulatory proteins in the apoptosis pathway in cytoskeletal movement, regulating cell growth, changes. Determining the mechanism underlying cisplatin forming ion channels, and participating in cellular signal resistance is of great significance to reverse the clinical transduction, among others (Mussunoor and Murray, 2008; tolerance and improve the 5-year survival rate of patients. Fatimathas and Moss, 2010). In recent years, studies have A1, A2, and A3 form intracellular membrane globules revealed that partial molecules enjoy close relationships that participate in calcium-dependent endocytosis and with cancer emergence and development, as well as drug exocytosis. By contrast, cisplatin resistance mainly resistance (Gerke et al., 2005; Chen et al., 2012). reduces the drug intake of cells and increases active efflux. Compared with that of corresponding normal The experiment proves that the expression of A1, A2, and tissues, A1 enjoys significant differences in expression A3 increases significantly in cisplatin-resistant A549 cells, in precancerous lesions of various tissues and tumors. which indicates that these molecules participate in drug In addition, a large number of studies indicated that A1 resistance in adenocarcinoma (Figureure 1). If A1, A2, does not consistent function in the development of drug- and A3 are specifically inhibited, the efflux and tolerance resistance in various tumors (Cao et al., 2008; Yu et al., of cancer cells towards medications such as cisplatin 2008;Wang et al., 2010). High A1 expression increases may be reduced, thereby enabling their accumulation drug resistance of breast adenocarcinoma; however, in in cancer cells, reaching their effective concentrations, NUGC cells, A1 deficiency is related to tumorigenesis and ultimately inducing cancer apoptosis. Consequently, and the development of resistance to the apoptosis induced compound molecule libraries were simulated and by chemotherapeutic agents. Other studies have proven established, and small molecules that combine with A1, that A1 participates in the formation of intracellular A2, and A3 were specifically screened (Figureure 2). multivesicle endosomes (Mussunoor and Murray, 2008). The paper studies the influence of A1, A2, and A3 However, whether the multi-vesicle endosome participates expression on cisplatin resistance in lung adenocarcinoma in the transport of certain chemotherapeutic agents within in vitro and in vivo, and new content on the emergence tumor cells has not been proven thus far. Meanwhile, and development drug resistance in lung adenocarcinoma A2 is abnormally expressed in tumors, such as gastric is added. Meanwhile, A1, A2, and A3 were specifically cancer, colon cancer, prostate cancer, HCC, and pancreatic targeted using small molecules to find new candidate Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 3193 Chao Wang et al 4-amino-2-tri-fluoromethyl-phenyl ester inhibition of cell compounds that effectively reverse drug resistance in growth and migration in the A549 lung adenocarcinoma cell adenocarcinoma, and provide more effective candidate line. Asian Pac J Cancer Prev, 14, 7265-70. treatment targets and drugs in the clinical treatment of Yan X, Yin J, Yao H, et al (2010). Increased expression of lung adenocarcinoma. annexin A3 is a mechanism of platinum resistance in ovarian cancer. Cancer Res, 70, 1616-24. Acknowledgements Yee DS, Narula N, Ramzy I, et al (2007). Reduced annexin II protein expression in high-grade prostatic intraepithelial This research is supported by the National Natural neoplasia and prostate cancer. Arch Pathol Lab Med, 131, Science Foundation of China (No. 81201985). 902-8. Yu G, Wang J, Chen Y, et al (2008). Tissue microarray analysis reveals strong clinical evidence for a close association References between loss of annexin A1 expression and nodal metastasis in gastric cancer. Clin Exp Metastasis, 25, 695-702. Cao Y, Li Y, Edelweiss M, et al (2008). Loss of annexin Zhang MC, Hu CP (2006). Lung cancer molecular mechanisms A1 expression in breast cancer progression. Appl of cisplatin resistance. Int J Respiration, 2, 152-5. Immunohistochem Mol Morphol, 16, 530-4. Zhou YM, Liu J, Sun W (2014). MiR-130a overcomes gefitinib Chen CY, Shen JQ, Wang F, et al (2012). Prognostic resistance by targeting met in non-small cell lung cancer cell Significance of Annexin A1 Expression in Pancreatic Ductal lines. Asian Pac J Cancer Prev, 15, 1391-6. Adenocarcinoma. Asian Pac J Cancer Prev, 13, 4707-12. Emoto K, Sawada H, Yamada Y, et al (2001). Annexin II over- expression is correlated with poor prognosis in human gastric carcinoma. Anticancer Res, 21, 1339-45. Esposito I, Penzel R, Chaib-Harrireche M, et al (2006). Tenascin C and annexin 2 expression in the process of pancreatic carcinogenesis. J Pathol, 208, 673-85. Fan Jiang, Jiang Ge-nin (2008). Advances in treatment of non- small cell lung cancer. Chinese J Clin, 2, 11-4. Fatimathas L, Moss SE (2010). Annexins as disease modifiers. Histol Histopathol, 25, 527-32. 2+ Gerke V, Creutz CE, Moss SE (2005). Annexins: linking Ca signalling to membrane dynamics. Nat Rev Mol Cell Biol, 6, 449-61. Gillette JM, Chan DC, Nielsen-Preiss SM (2004). Annexin 2 expression is reduced in human osteosarcoma metastases. J Cell Biochem, 92, 820-32. Huang Y, Jin Y, Yan CH, et al (2008). Involvement of Annexin A2 in p53 induced apoptosis in lung cancer. Mol Cell Biochem, 309, 117-23. Langer CJ, Besse B, Gualberto A, et al (2010). The evolving role of histology in the management of advanced non-small-cell lung cancer. J Clin Oncol, 28, 5311-20. Mussunoor S, Murray GI (2008). The role of annexins in tumour development and progression. J Pathol, 216, 131-40. Protzel C, Richter M, Poetsch M, et al (2011). The role of annexins I, II and IV in tumor development, progression and metastasis of human penile squamous cell carcinomas. World J Urol, 29, 393-8. Sharma MR, Koltowski L, Ownbey RT, et al (2006). Angiogenesis-associated protein annexin II in breast cancer: selective expression in invasive breast cancer and contribution to tumor invasion and progression. Exp Mol Pathol, 81, 146-56. Stewart DJ, Chiritescu G, Dahrouge S, et al (2007). Chemotherapy dose--response relationships in non-small cell lung cancer and implied resistance mechanisms. Cancer Treat Rev, 33, 101-37. Stewart DJ (2010). Tumor and host factors that may limit efficacy of chemotherapy in non-small cell and small cell lung cancer. Crit Rev Oncol Hematol, 75, 173-234. Sun DS, Hu LK, Cai Y, et al (2014). A systematic review of risk factors for brain metastases and value of prophylactic cranial irradiation in non-small cell lung cancer. Asian Pac J Cancer Prev, 15, 1233-9. Wang LP, Bi J, Yao C, et al (2010). Annexin A1 expression and its prognostic significance in human breast cancer. Neoplasma, 57, 253-9. Wang H, Gui SY, Chen FH, et al (2013). New insights into 3194 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Asian Pacific Journal of Cancer Prevention Unpaywall

Regulatory Mechanisms of Annexin-Induced Chemotherapy Resistance in Cisplatin Resistant Lung Adenocarcinoma

Wang, Chao; Xiao, Qian; Li, Yu-Wen; Zhao, Chao; Jia, Na; Li, Rui-Li; Cao, Shan-Shan; Cui, Jia; Wang, Lu; Wu, Yin; Wen, Ai-Dong
Asian Pacific Journal of Cancer Prevention – Apr 1, 2014
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1513-7368
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10.7314/apjcp.2014.15.7.3191
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Abstract

Adenocarcinoma of lung has high incidence and a poor prognosis, woith chemotherapy as the main therapeutic tool, most commonly with cisplatin. However, chemotherapy resistance develops in the majority of patients during clinic treatment. Mechanisms of resistance are complex and still unclear. Although annexin play important roles in various tumor resistance mechanisms, their actions in cisplatin-resistant lung adenocarcinoma remain unclear. Preliminary studies by our group found that in cisplatin-resistant lung cancer A549 cells and lung adenocarcinoma tissues, both mRNA and protein expression of annexins A1, A2 and A3 is increased. Using a library of annexin A1, A2 and A3 targeting combined molecules already established by ourselves we found that specific targeting decreased cisplatin-resistance. Taken together, the underlined effects of annexins A1, A2 and A3 on drug resistance and suggest molecular mechanisms in cisplatin-resistant A549 cells both in vivo and in vitro. Furthermore, the study points to improved research on occurrence and development of lung adenocarcinoma, with provision of effective targets and programmes for lung adenocarcinoma therapy in the clinic. Keywords: Annexin - lung adenocarcinoma - chemotherapy - effective targets Asian Pac J Cancer Prev, 15 (7), 3191-3194 studies indicate that Annexins (A1, A2, and A3) play Introduction significant roles in the growth of various tumors and the Lung cancer, with its rapidly growing morbidity, is production of drug resistance, which mark good target one of the common malignancies that ranks among the spots for tumor treatment (Mussunoor and Murray, 2008; top in the incidence of cancer among adults, and has Protzel et al., 2010). become the most common cause of human death from However, reports on the functions and mechanisms of tumors (Sun et al., 2014). Adenocarcinoma accounts action of Annexins (A1, A2, and A3) on adenocarcinoma for 40% of lung cancer incidence (Langer et al., 2010). and cisplatin-resistance have not been published. This Adenocarcinoma cannot be treated adequately through experiment aims to study the mechanisms of Annexins simple operations. Therefore, chemotherapy remains (A1, A2, and A3) in inhibiting cisplatin induced the mainstay treatments for lung cancer (Wang et al., apoptosis of adenocarcinoma cells, and primarily screen 2013; Zhou et al., 2014). Cisplatin, as the first line drug, and construct compound molecule libraries that can be is extensively applied for the clinical treatment of non- combined with Annexins (A1, A2, and A3)through crystal small-cell lung cancer (Fan and Jiang, 2008). However, diffraction and molecular space simulation. the drug resistance produced by tumor cells marks not only the most universal and difficult problem that influences Materials and Methods the curative effect of chemotherapy, but also the main reason that leads to chemotherapy failure. However, Cell culture, DDP resistant A549 stable cell line and no therapeutic schedule or drug effectively inhibits the primary cell culture from lung adenocarcinoma patients tolerance of adenocarcinoma and extend the life of patients A549 cells were maintained in RPMI supplemented have been developed so far (Zhang and Hu, 2006; Stewart with 10% fetal bovine serum, 2 mmol/l of glutamine, 100 et al., 2007; Stewart, 2010). Therefore, conducting in- units/ml of penicillin and 100 mg/ml of streptomycin in depth research on the new action mechanism of cisplatin a 5% CO atmosphere at 37°C. A549 cells were treated on adenocarcinoma and discovering key molecules that with DDP (1 ug/ml) for 48h, once a month, 6 times. A549/ play significant roles may lead to more effective candidate DDP cells resistant index for DDP is 18, meanwhile, with targets and treatment plans for adenocarcinoma. Many cross resistance to Carboplatin. 1 2 & Department of Pharmacy, Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China Equal contributors *For correspondence: [email protected], [email protected] Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 3191 Chao Wang et al RT-PCR therapy for cisplatin resistance. Therefore, screening Expression of AnnexinA1, A2, A3 were analyzed by through Crystallography and Molecular space simulation, semiquantitative RT-PCR analysis. After the total RNA we preliminarily constructed libraries of molecular was prepared using a TRIZOL reagent (Life Technologies, compounds which could bind with Annexin A1, A2 and Inc., 15596-026), the oligo (dT)-primed cDNA was A3 respectively. Three compounds: compound1, 2 and 3 synthesized using a RT-PCR kit (Agilent Technologies, which binding with Annexin A1, A2 and A3 respectively 600182). The primers used in this study were summarized were selected and the structures are shown here (Figure in Table 1. 3). Annexin A1, A2 and A3 are all small molecule compounds, both A1 and A3 contain a benzo seven- Immunoblot analysis membered-ring. A2 contains a benzo five-membered- After treatment with different conditions as described ring, A1 and A3 contain an amide bond with α-amino. in the figure legends, cells were lysed in M2 buffer (20 Treatment of A549/DDP cells with compound 1, 2 and mM Tris at pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM 3, indeed, A549/DDP cells viability were dramatically EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 20 decreased (Figure 4). The result confirmed effectiveness of mM β-glycerol phosphate, 1 mM sodium vanadate, 1 mg/ ml leupeptin). Fifty micrograms of the cell lysates were subjected to SDS-polyacrylamide gel and blotted onto PVDF membrane (Millipore, PVH00010). After blocking with 5% skim milk in TBS/T, the membrane was probed with the relevant antibody and visualized by enhanced chemiluminescence (ECL, RPN2106), according to the manufacturer’s instruction (Amersham). Immunofluorescence analysis HeLa cells were grown on glass coverslips and then infected with Ad-GFP-LC3. After 24h, the cells were treated as indicated in the figure legend and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with PBS containing 0.2% Triton X-100 and 0.1 M glycine for 15 min, blocked with 10%BSA in PBS for 1 h and then incubated with rat anti-human Figureure 1. High Expression of Annexin A1, A2 and Statistical analysis A3 in A549/DDP Cells. mRNA expression of Annexin A1,A2 Data are expressed as the mean±SD from at least three and A3 in A549 and A549/DDP cells (left). Relative intensity of band were analyzed (right); Protein expression of Annexin A1, separate experiments performed triplicate. The differences A2, A3 in A549 and A549/DDP cells (left). Relative intensity between groups were analyzed using Student’s t-test. of band were analyzed (right) p<0.05 is considered statistically significant. Statistical analyses were performed using SPSS software ver. 13.0 (SPSS, Inc.). Results Firstly, in order to identify involvement of Annexins in cisplatin resistance, we constructed DDP resistant A549 lung adenocarcinoma stable cell line (A549/DDP), and compared expression of Annexin A1, A2 and A3 to A549 and A549/DDP in both mRNA and protein levels. Annexin A1, A2 and A3 highly expressed in A549/DDP cells (Figure 1 A, B), which suggested expression of Annexin A1, A2 and A3 closely related to cisplatin resistance. To further reveal the critical role of Annexin A1, A2 and A3 in cisplatin resistance in vivo, we observed the expression of Annexin A1, A2 and A3 in tumor tissues of lung adenocarcinoma patients. In consistent with in vitro data, expression of Annexin A1, A2 and A3 were up-regulated Figureure 2. High Expression of Annexin A1, A2 and in cisplatin resistant patients’ tissues both in mRNA and A3 in Cisplatin Resistant Cells From Tumors of Lung protein levels (Figure 2). Taken together, Annexin A1, A2 Adenocarcinoma Patients. mRNA protein expression and A3 overexpressed both in vitro and in vivo, suggested of Annexin A1, A2 and A3 in cisplatin resistant cells from they are most likely to be key regulator of cisplatin tumors of lung adenocarcinoma patients (lane1,3,5) and non- resistance, thus pharmacological approaches targeting cisplatin resistant cells from tumors of another part of lung to Annexin A1, A2 and A3 are considered as effective adenocarcinoma patients (lane2,4,6). 3192 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.7.3191 Annexin-Induced Chemotherapy Resistance in Cisplatin Resistant Lung Adenocarcinoma ductal adenocarcinoma, and is related to tumor invasion, metastasis, and prognosis. Moreover, previous studies reported that high A2 expression is related to the degree of malignancy, as well as invasion and metastasis (Emoto et al., 2001; Esposito et al., 2006; Sharma et al., 2006; Yee et al., 2007; Duncan et al, 2008). A2 is selectively Figureure 3. Hypothetic Molecular Structures of expressed in lung adenocarcinoma cell lines. Furthermore, Annexins Binding Compounds. Screening and construction of Annexin A1, A2 and A3 binding compounds libraries A2 promotes the invasion and metastasis of lung cancer respectively through Crystallography and Molecular space cells. However, no statement has been made regarding its simulation (ACCELRYS DISCOVERY STUDIO V1.6) relationship with cisplatin-resistance in adenocarcinoma (Gillette et al., 2004; Huang et al., 2008). Meanwhile, recent research results indicated that A3 expression in the tissues of patients with cisplatin-resistant prostate cancer and ovarian cancer has increased significantly. In addition, the cycles of patients without tumors in the A3 high expression group are clearly shortened (Gillette et al., 2004; Huang et al., 2008) Related studies also prove that A3 produces drug resistance by reducing the platinum content, the platinum-DNA combining quantity within cells, and the p53 level in ovarian cancer cells (Yan et al., Figureure 4. Effect of Annexins Binding Compounds 2010). The tumorigenesis of cells with high A3 expression on A549/DDP Cells. A549/DDP cells were treated with is significantly increased after cisplatin treatment, as compound1 (10 ug/ml), compound2 (10 ug/ml), compound3 demonstrated through an animal experiment. In addition, (10 ug/ml) vevfrom libraries respectively, cell viability was the resistance of cells with high A3 expression is clearly determined with MTT enhanced against platinum-based medicines; however, it Annexin A1, A2 and A3 targeting compounds in inducing does not induce cross-resistance with TAX and EPI (Yan cisplatin resistant A549 cell death. Furthermore, our study et al., 2010). provided powerful possibility of the concept of targeting Platinum resistance mechanisms, which are still the Annexin A1, A2 and A3 for the treatment of cisplatin unclear, mainly involve the following aspects: 1. Reduced resistant lung adenocarcinoma patients drug accumulation within tumor cells, including reducing cellular drug intake and increasing active efflux; 2. Functions of tumor cells to withstand the transformation Discussion of cancer medicines, as well as enhanced detoxification; The annexin family is super family of calphobindin, that 3. Strengthened DNA repair by tumor cells; 4. In the present in the intracellularly among major eucaryons, and induction of apoptosis by platinum-based medicines, the play crucial physiologic functions, such as participating expression of regulatory proteins in the apoptosis pathway in cytoskeletal movement, regulating cell growth, changes. Determining the mechanism underlying cisplatin forming ion channels, and participating in cellular signal resistance is of great significance to reverse the clinical transduction, among others (Mussunoor and Murray, 2008; tolerance and improve the 5-year survival rate of patients. Fatimathas and Moss, 2010). In recent years, studies have A1, A2, and A3 form intracellular membrane globules revealed that partial molecules enjoy close relationships that participate in calcium-dependent endocytosis and with cancer emergence and development, as well as drug exocytosis. By contrast, cisplatin resistance mainly resistance (Gerke et al., 2005; Chen et al., 2012). reduces the drug intake of cells and increases active efflux. Compared with that of corresponding normal The experiment proves that the expression of A1, A2, and tissues, A1 enjoys significant differences in expression A3 increases significantly in cisplatin-resistant A549 cells, in precancerous lesions of various tissues and tumors. which indicates that these molecules participate in drug In addition, a large number of studies indicated that A1 resistance in adenocarcinoma (Figureure 1). If A1, A2, does not consistent function in the development of drug- and A3 are specifically inhibited, the efflux and tolerance resistance in various tumors (Cao et al., 2008; Yu et al., of cancer cells towards medications such as cisplatin 2008;Wang et al., 2010). High A1 expression increases may be reduced, thereby enabling their accumulation drug resistance of breast adenocarcinoma; however, in in cancer cells, reaching their effective concentrations, NUGC cells, A1 deficiency is related to tumorigenesis and ultimately inducing cancer apoptosis. Consequently, and the development of resistance to the apoptosis induced compound molecule libraries were simulated and by chemotherapeutic agents. Other studies have proven established, and small molecules that combine with A1, that A1 participates in the formation of intracellular A2, and A3 were specifically screened (Figureure 2). multivesicle endosomes (Mussunoor and Murray, 2008). The paper studies the influence of A1, A2, and A3 However, whether the multi-vesicle endosome participates expression on cisplatin resistance in lung adenocarcinoma in the transport of certain chemotherapeutic agents within in vitro and in vivo, and new content on the emergence tumor cells has not been proven thus far. Meanwhile, and development drug resistance in lung adenocarcinoma A2 is abnormally expressed in tumors, such as gastric is added. Meanwhile, A1, A2, and A3 were specifically cancer, colon cancer, prostate cancer, HCC, and pancreatic targeted using small molecules to find new candidate Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 3193 Chao Wang et al 4-amino-2-tri-fluoromethyl-phenyl ester inhibition of cell compounds that effectively reverse drug resistance in growth and migration in the A549 lung adenocarcinoma cell adenocarcinoma, and provide more effective candidate line. Asian Pac J Cancer Prev, 14, 7265-70. treatment targets and drugs in the clinical treatment of Yan X, Yin J, Yao H, et al (2010). Increased expression of lung adenocarcinoma. annexin A3 is a mechanism of platinum resistance in ovarian cancer. Cancer Res, 70, 1616-24. Acknowledgements Yee DS, Narula N, Ramzy I, et al (2007). Reduced annexin II protein expression in high-grade prostatic intraepithelial This research is supported by the National Natural neoplasia and prostate cancer. Arch Pathol Lab Med, 131, Science Foundation of China (No. 81201985). 902-8. Yu G, Wang J, Chen Y, et al (2008). Tissue microarray analysis reveals strong clinical evidence for a close association References between loss of annexin A1 expression and nodal metastasis in gastric cancer. Clin Exp Metastasis, 25, 695-702. Cao Y, Li Y, Edelweiss M, et al (2008). Loss of annexin Zhang MC, Hu CP (2006). Lung cancer molecular mechanisms A1 expression in breast cancer progression. Appl of cisplatin resistance. Int J Respiration, 2, 152-5. Immunohistochem Mol Morphol, 16, 530-4. Zhou YM, Liu J, Sun W (2014). MiR-130a overcomes gefitinib Chen CY, Shen JQ, Wang F, et al (2012). Prognostic resistance by targeting met in non-small cell lung cancer cell Significance of Annexin A1 Expression in Pancreatic Ductal lines. 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Published: Apr 1, 2014

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