2010-01-012025-05-02https://scholars.lib.ntu.edu.tw/handle/123456789/728768肺癌是致死率極高的癌症之一。肺癌的分類以非小細胞癌為主，包括腺癌，肺泡細胞癌，及扁平細胞癌。肺癌的治療有手術、化療與放射線療法。一般而言，接受化療的肺癌病人可增加平均一年的存活率。肺癌的高死亡率在於早期肺癌沒有症狀，一旦有嚴重咳嗽，咳血，胸痛，及呼吸困難時，已經是第三或第四期，錯失手術切除的時機，預後自然十分不佳，五年存活率只有5-10%。先前的研究指出，表達較低量的修補蛋白ERCC1的肺癌患者，其對於化學治療效果較佳。此外，修補蛋白Rad51表現量與癌細胞對化學治療與放射治療產生抗性有關。因此，降低細胞內ERCC1或Rad51的表現可以增加化療藥物的敏感性。 大黃素(emodin)為天然的活性物質，常存在於中草藥中例如虎丈的根部與何首烏。文獻指出大黃素具有毒殺肺癌細胞的能力。大黃素可在癌細胞中誘發細胞週期暫停和細胞程序性凋亡。文獻指出大黃素可抑制ERK1/2的活化。而我們先前的研究成果發現ERK1/2訊號路徑調控修補蛋白Rad51表現。因此，大黃素可能經由抑制ERK1/2訊號而壓制Rad51和ERCC1蛋白質的含量，降低細胞內核酸修補能力，進而毒殺肺癌細胞。因此本實驗目的，在探討不同的非小細胞肺癌在大黃素處理下，細胞內ERK1/2激酶與ERCC1或Rad51蛋白的活性與表現量之變化。同時評估若同時壓制ERCC1或Rad51蛋白質的表現對於大黃素誘發肺癌細胞毒性，細胞凋亡，細胞增生等是否有相加成之效果。希望本實驗在未來肺癌的治療上能提供更好的方法與研究方向，並且在天然中草藥活性物質治療肺癌有更進一步的了解。Emodin Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a naturally occurring anthraquinone present in the roots and rhizomes of numerous plants and Chinese herbs (Demirezer et al., 2001; Kuo et al., 2001). A number of studies have demonstrated that emodin is capable of inducing cell apoptosis and growth arrest in various cancer cells, such as human lung cancer cells (Su et al., 2005), HER-2/neu–overexpressed breast cancer cells (Zhang et al., 1998; Zhang et al., 1999), cervical cancer cells, leukemia cells, hepatoma cells, and prostate cancer cell lines (Chen et al., 2002; Shieh et al., 2004; Wang et al., 2007). In addition, emodin induces sub-G1 accumulation and G2/M phase arrest in hepatoma cells (Shieh et al., 2004). Emodin, as a tyrosine kinase inhibitor, can inhibit the kinase activity of HER-2/neu and suppress the proliferation of HER-2/neu–overexpressed non-small cell lung cancer (NSCLC) and breast cancer cells (Zhang et al., 1995; Zhang et al., 1999). Emodin has also been shown to inhibit p56lck (Zhang et al., 1995) and casein kinase 2 (CK2) activity (Battistutta et al., 2000). In addition, emodin can repress epidermal growth factor receptor (EGFR) tyrosine kinase activity at high concentrations (Zhang et al., 1999). The molecular mechanism by which emodin induces cell apoptosis is through inactivation of ERK and AKT and the decrease of anti-apoptotic protein Bcl2 levels in human A549 cells (Su et al., 2005). However, the effect of emodin on the ability of DNA repair to regulate cell survival in NSCLC cells has not yet been fully defined. Excision repair cross-complementary 1 (ERCC1) Excision repair cross-complementary 1 (ERCC1) has a leading role in the nucleotide excision repair (NER) process because of its involvement in the excision of DNA adducts (de Laat et al., 1998). The damaged DNA strand is cleaved by ERCC1-XPF (xeroderma pigmentosum-F) on the 5' side during NER in human cells (Evans et al., 1997). ERCC1 RNA levels are highly correlated with NER activity in blood lymphocytes (Vogel et al., 2000). The increased expression of ERCC1 is associated with clinical resistance to platinum-based chemotherapy in human NSCLC (Lord et al., 2002). Moreover, patients with ERCC1-negative tumors had a longer survival time than those with ERCC1-positive tumors in NSCLC (Olaussen et al., 2006). Rad51 Homologous recombination repair (HRR), a major pathway for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells, is mediated by Rad51. The HRR process consists of assembly of Rad51 onto DNA substrates at the site of a DNA break to form a helical nucleoprotein filament, which catalyzes homologous pairing and joins in a hetero-duplex formation with the sister chromatid (West, 2003). Rad51 is overexpressed in many tumors (Raderschall et al., 2002; Xia et al., 1997), and its overexpression is related to the resistance of the tumor to chemotherapeutic agents or radiation (Bello et al., 2002; Slupianek et al., 2002). In NSCLC, high expression of Rad51 in tumor tissue is associated with an unfavorable prognosis (Qiao et al., 2005; Takenaka et al., 2007). On the other hand, inhibition of Rad51 expression has been shown to sensitize cancer cells to radiotherapy and chemotherapeutic agents (Ko et al., 2008a; Ohnishi et al., 1998). However, whether Rad51 and ERCC1 are targeted by emodin to induce cell death in human lung cancer cells is still unclear and remains to be determined. MKK1/2-ERK1/2 signaling pathway ERK1/2 is serine-threonine protein kinase and one of mitogen-activated protein kinases (MAPKs) (Chang and Karin, 2001; Garrington and Johnson, 1999; Kolch, 2000; Kolch, 2005; Nebreda and Porras, 2000). The ERK1/2 signaling activation are connected to cell surface receptors, such as EGFR and platelet-derived growth factor receptor (PDGFR), and activated via interaction with a family of small GTPases, such as Ras (Kolch, 2000). In general, ERK1/2 is involved in the control of cell proliferation, survival, differentiation, growth arrest, apoptosis, cell transformation and tumor invasion through phosphorylation of many downstream substrates including transcription factors, intracellular protein kinases, phosphatases and cytoskeletal proteins (Kolch, 2000; Kolch, 2005; Marshall, 1995). In addition, ERK1/2 signaling pathway activation plays a vital role in tumorigenesis (Weinstein-Oppenheimer et al., 2000) and promotes tumor cell growth in several cancer cell lines (Albanell et al., 2001; El Sheikh et al., 2004). Rationale Lung cancer remains the leading cause of cancer-related deaths in the world, and more than 85% of lung cancers have NSCLC (Landis et al., 1999). In this study, we investigated the role of emodin in suppressing cell viability in four NSCLC cell lines and examined possible molecular mechanisms for this activity. We also determine the roles of repair proteins ERCC1 and Rad51 in emodin-induced cytotoxicity. Emodin may be a novel and improved therapeutic modality for advanced lung cancer in the future, especially for patients in whom lung cancer cells are resistant to chemotherapeutic agents. Emodin Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a naturally occurring anthraquinone present in the roots and rhizomes of numerous plants and Chinese herbs (Demirezer et al., 2001; Kuo et al., 2001). A number of studies have demonstrated that emodin is capable of inducing cell apoptosis and growth arrest in various cancer cells, such as human lung cancer cells (Su et al., 2005), HER-2/neu–overexpressed breast cancer cells (Zhang et al., 1998; Zhang et al., 1999), cervical cancer cells, leukemia cells, hepatoma cells, and prostate cancer cell lines (Chen et al., 2002; Shieh et al., 2004; Wang et al., 2007). In addition, emodin induces sub-G1 accumulation and G2/M phase arrest in hepatoma cells (Shieh et al., 2004). Emodin, as a tyrosine kinase inhibitor, can inhibit the kinase activity of HER-2/neu and suppress the proliferation of HER-2/neu–overexpressed non-small cell lung cancer (NSCLC) and breast cancer cells (Zhang et al., 1995; Zhang et al., 1999). Emodin has also been shown to inhibit p56lck (Zhang et al., 1995) and casein kinase 2 (CK2) activity (Battistutta et al., 2000). In addition, emodin can repress epidermal growth factor receptor (EGFR) tyrosine kinase activity at high concentrations (Zhang et al., 1999). The molecular mechanism by which emodin induces cell apoptosis is through inactivation of ERK and AKT and the decrease of anti-apoptotic protein Bcl2 levels in human A549 cells (Su et al., 2005). However, the effect of emodin on the ability of DNA repair to regulate cell survival in NSCLC cells has not yet been fully defined. Excision repair cross-complementary 1 (ERCC1) Excision repair cross-complementary 1 (ERCC1) has a leading role in the nucleotide excision repair (NER) process because of its involvement in the excision of DNA adducts (de Laat et al., 1998). The damaged DNA strand is cleaved by ERCC1-XPF (xeroderma pigmentosum-F) on the 5' side during NER in human cells (Evans et al., 1997). ERCC1 RNA levels are highly correlated with NER activity in blood lymphocytes (Vogel et al., 2000). The increased expression of ERCC1 is associated with clinical resistance to platinum-based chemotherapy in human NSCLC (Lord et al., 2002). Moreover, patients with ERCC1-negative tumors had a longer survival time than those with ERCC1-positive tumors in NSCLC (Olaussen et al., 2006). Rad51 Homologous recombination repair (HRR), a major pathway for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells, is mediated by Rad51. The HRR process consists of assembly of Rad51 onto DNA substrates at the site of a DNA break to form a helical nucleoprotein filament, which catalyzes homologous pairing and joins in a hetero-duplex formation with the sister chromatid (West, 2003). Rad51 is overexpressed in many tumors (Raderschall et al., 2002; Xia et al., 1997), and its overexpression is related to the resistance of the tumor to chemotherapeutic agents or radiation (Bello et al., 2002; Slupianek et al., 2002). In NSCLC, high expression of Rad51 in tumor tissue is associated with an unfavorable prognosis (Qiao et al., 2005; Takenaka et al., 2007). On the other hand, inhibition of Rad51 expression has been shown to sensitize cancer cells to radiotherapy and chemotherapeutic agents (Ko et al., 2008a; Ohnishi et al., 1998). However, whether Rad51 and ERCC1 are targeted by emodin to induce cell death in human lung cancer cells is still unclear and remains to be determined. MKK1/2-ERK1/2 signaling pathway ERK1/2 is serine-threonine protein kinase and one of mitogen-activated protein kinases (MAPKs) (Chang and Karin, 2001; Garrington and Johnson, 1999; Kolch, 2000; Kolch, 2005; Nebreda and Porras, 2000). The ERK1/2 signaling activation are connected to cell surface receptors, such as EGFR and platelet-derived growth factor receptor (PDGFR), and activated via interaction with a family of small GTPases, such as Ras (Kolch, 2000). In general, ERK1/2 is involved in the control of cell proliferation, survival, differentiation, growth arrest, apoptosis, cell transformation and tumor invasion through phosphorylation of many downstream substrates including transcription factors, intracellular protein kinases, phosphatases and cytoskeletal proteins (Kolch, 2000; Kolch, 2005; Marshall, 1995). In addition, ERK1/2 signaling pathway activation plays a vital role in tumorigenesis (Weinstein-Oppenheimer et al., 2000) and promotes tumor cell growth in several cancer cell lines (Albanell et al., 2001; El Sheikh et al., 2004). Rationale Lung cancer remains the leading cause of cancer-related deaths in the world, and more than 85% of lung cancers have NSCLC (Landis et al., 1999). In this study, we investigated the role of emodin in suppressing cell viability in four NSCLC cell lines and examined possible molecular mechanisms for this activity. We also determine the roles of repair proteins ERCC1 and Rad51 in emodin-induced cytotoxicity. Emodin may be a novel and improved therapeutic modality for advanced lung cancer in the future, especially for patients in whom lung cancer cells are resistant to chemotherapeutic agents.非小細胞肺癌大黃素ERCC1修補蛋白質Rad51修補蛋白質核酸修補細胞毒性