CANCER BIOTHERAPY AND RADIOPHARMACEUTICALS Volume 30, Number 2, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/cbr.2014.1797
DNA Methylation of DKK3 Modulates Docetaxel Chemoresistance in Human Nonsmall Cell Lung Cancer Cell Leilei Tao, Guichun Huang, Yitian Chen, and Longbang Chen
Abstract
Dickkopf-related protein 3 (DKK3) gene, as a tumor suppressor gene, has been discovered in various cancers, but its relationship with tumor chemoresistance is still unclear. In this study, this laboratory detected that DNA methylation contributes to the downregulation of DKK3 in docetaxel resistance of human lung cancer cells and its possible biochemical mechanism. DKK3 has been proved to be downregulated by hypermethylation in docetaxelresistant lung cancer cells. Upregulation of DKK3 can reverse the chemoresistance of docetaxel-resistant cell lines in vitro by growth inhibition and enhancement of apoptosis. Conversely, downregulation of DKK3 could induce parental human lung cancer cells insensitivity to docetaxel by promoting proliferative capacity and inhibiting apoptosis of cancer cells. In addition, the authors observed that overexpression of DKK3 might decrease the expression of P-glycoprotein. All results suggested that epigenetic downregulation of DKK3 leads to docetaxel resistance in human nonsmall cell lung cancer (NSCLC) cells by increased expression of P-glycoprotein. DKK3 may reveal a novel molecular target for docetaxel resistance for NSCLC patients in the future. Key words: chemoresistance, DKK3, DNA methylation, docetaxel, NSCLC Introduction
L
ung cancer is one of the highest incidence of malignancies and leading cause of cancer-related death in the world. It is reported that 80% of all lung cancer patients are nonsmall cell lung cancer (NSCLC). Most of the lung cancer patients are diagnosed in the advanced stages (Stages III and IV).1 Despite development of cancer treatment and introduction of new technology, advanced lung cancer remains associated with poor prognosis. Docetaxel, a semisynthetic analogue of paclitaxel, is widely used as an anticancer drug in the treatment of lung cancer.2 Docetaxel confers its antineoplastic activity by inhibiting microtubule depolymerization, leading the metaphase to anaphase transition arrest, activating the spindle assembly checkpoint, and subsequently causing apoptosis.3,4 However, chemotherapy agents become increasingly ineffective when the disease has spread beyond the lung at the advanced stage. Chemoresistance almost occurs in recurrent patients and remains the most important dilemma in restricting the clinical application of anticancer drugs.5 Thus, a further study of the molecular mechanism underlying
docetaxel resistance of NSCLC should be helpful to improve the efficacy of its clinical application. Accumulated evidences have proved that cancer cells resistant to docetaxel result from both genetic and epigenetic modification of various critical genes.6,7 Aberrant DNA methylation, leading to downregulation of key genes, which played a crucial role during the initiation and development of cancer.8 However, the correlation between DNA methylation and docetaxel resistance of NSCLC is rarely reported. In the laboratory, the authors previously established a docetaxel-resistant H1299 cell line (H1299/DTX) by continuous exposure to increasing concentrations of docetaxel. It is important for us to understand the molecular mechanisms underlying docetaxel resistance and explore novel therapeutic markers for reversing chemoresistance in NSCLC. Dickkopf-related protein 3 (DKK3), a putative Wnt antagonist, has been proved to represent as a tumor suppressor for downregulation in various human cancers. In many types of cancers, the failure of its normal expression is closely associated to CpG island methylation on the DKK3 promoter; thus, the published articles of DKK3 have mainly
Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University, Nanjing, People’s Republic of China. Address correspondence to: Longbang Chen; Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University; 305 ZhongShan Eastern Road, Nanjing 210002, People’s Republic of China. E-mail: [email protected]
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DKK3 AND LUNG CANCER CHEMORESISTANCE
focused on the methylation-related field.9 The silencing expression of DKK3 is always correlated with poor prognosis of human cancer, including lung cancer,10 ovarian cancer,11 renal clear cell carcinoma,12 and prostate cancer.13 However, the association of DKK3 with chemotherapy resistance in NSCLC cells was seldom seen in the previous studies. It is vitally necessary that the molecular mechanism underlying docetaxel resistance should be elucidated and searched for novel therapeutic markers for reversing chemoresistance. In this study, the authors investigated that epigenetic downregulation of DKK3 mediated by aberrant promoter methylation contributes to docetaxel resistance in NSCLC cell lines. At the same time, DKK3 re-expression could reverse docetaxel resistance of lung cancer cell. The results of this study may provide a novel strategy for reversing chemoresistance of NSCLC. Materials and Methods Cell culture
Human NSCLC H1299 cells were bought from the Cell Bank of Chinese Academy of Medical Science. Cells were conditioned in the RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/L streptomycin in a humidified atmosphere at 37C, 5% CO2. The docetaxel-resistant H1299 cell line (H1299/DTX) was established by continuous exposure to increasing concentrations of docetaxel, and cells were grown in the presence of 5 lg/L final concentration of docetaxel. RNA isolation and semiquantitative reverse transcription–polymerase chain reaction
Total RNA of tumor cells was isolated using the Trizol reagent (Invitrogen), and the spectrophotometer (BioPhotometer) was used to measure the concentration of total RNA. Reverse transcription (RT) was accomplished by the PrimeScript RT Reagent Kit (Takara) following the instruction manual of the manufacturer. Two micrograms of total RNA was applied for cDNA synthesis, and 20 lL reaction volume was prepared for further polymerase chain reaction (PCR). PCR conditions were in accordance with the manufacturer’s instructions. The primer sequences for each
101
gene were designed as shown in Table 1. The transcription of glyceraldehyde 3-phophate dehydrogenase (GAPDH) was performed for normalization. The PCR products were electrophoresed on 2% agarose gels and detected by ethidium bromide (EB) staining. Methylation-specific PCR
Methylation-specific PCR (MSP) was performed to detect the methylation status of DKK3 gene promoter by using bisulfate-modified genomic DNA as the template. Total DNA was isolated from cultured cells using the QIAamp DNA Mini Kit (Qiagen). One microgram of genomic DNA was bisulfate treated with the EZ-DNA methylation Gold Kit (Zymo Research) and finally resuspended in 10 lL TE buffer. Two sets of primers were used to display the differences between methylated and unmethylated status. The primers targeting the unmethylated and methylated DKK3 promoter regions are described in Table 1. The following steps were performed with an instruction manual. The PCR products were visualized in 2% agarose gel by EB staining. Lymphocyte DNA was used as unmethylation and methylation-positive control in the experiment. A water blank was performed as a negative control. Transient transfection plasmid or siRNA interference
The DKK3 expressing plasmid (pCS2-hDKK3) and negative control were available from Addgene (Plasmid 15496). Small interfering RNAs (siRNAs) targeting DKK3 (siRNA-DKK3) were obtained from Santa Cruz Biotechnology, Inc. Nonspecific control siRNA (siRNA/NC) was used as control. Plasmid DNA was extracted by the Plasmid Mini Kit (Qiagen). Cells were cultured in six-well plates (2 · 105cells/well) and transfected with the SiRNA Mate (GenePharma) or Turbofect Transfection reagent (Thermo Scientific) following the manufacturer’s protocol. Western blot
The cells were isolated directly or 72 hours after transfection; the cells were washed twice with phosphate buffered saline (PBS) before the proteins were extracted. Proteins were separated electrophoretically by 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene
Table 1. Primer Sequences Used for Quantitative PCR mRNA
Primers
DKK3
Forward: 5¢-GTTGAGGAACTGATGGAGGACA-3¢ Reverse: 5¢-TTGCACACATACACCAGGCTGT-3¢ Forward: 5¢-GGGGCGGGCGGCGGGGC-3¢ Reverse: 5¢-ACATCTCCGCTCTACGCCCG-3¢ Forward: 5¢-TTAGGGGTGGGTGGTGGGGT-3¢ Reverse: 5¢-CTACATCTCCACTCTACACCCA-3¢ Forward: 5¢-TGCGACAGGAGATAGGCTG-3¢ Reverse: 5¢-GCCAAAATCACAAGGGTTAGCTT-3¢ Forward: 5¢-GCACCGTCAAGGCTGAGAAC-3¢ Reverse: 5¢-TGGTGAAGACGCCAGTGGA-3¢
DKK3 (MSP-M) DKK3 (MSP-U) MDR-1 GAPDH
Expected size (bp) 695 118 126 193 138
PCR, polymerase chain reaction; DKK3, Dickkopf-related protein 3; MDR-1, multidrug resistance-1; GAPDH, glyceraldehyde 3phophate dehydrogenase.
102
fluoride (PVDF) membrane. The membranes were blocked for 2 hours with 5% nonfat milk at room temperature. The membranes were incubated first with the REIC/DKK3 antibody (1:2000; Abcam) or P-glycoprotein antibody (1:500; Bioss) overnight at 4C and then incubated with a second antibody for 2 hours at 37C. Proteins were then displayed with the ECL substrate (Cell Signaling Technology) in accordance with the manufacturer’s instructions. Protein levels were normalized to GAPDH. Cell viability assay
Single-cell suspensions were planted into 96-well plates (2 · 103 cells/well) directly or 24 hours after transfection. Various concentrations of docetaxel (0, 10, 20, 40, 80, 160, 320, 640, 1280 lg/L) were then added. Forty-eight hours later, cell viability was shown by the MTT assay. Colony formation assay
Three hundred cells were planted into each well in sixwell plates directly or 48 hours after transfection. After 14
TAO ET AL.
days, cells were fixed with methanol and stained with 0.1% crystal violet. Then, the number of visible colonies was counted. The cloning efficiency was calculated as follows: (the clone number/the number of plant cells) ·100%. Flow cytometric analysis of apoptosis
Cells were harvested 48 hours after transfection for cell apoptosis measurement, then resuspended in 0.5 mL binding buffer with 5 mL Annexin V and 5 mL propidium iodide (PI), and reacted for 15 minutes at 37C in the dark. Then, the early apoptotic rate was measured by flow cytometric analysis following the manufacturer’s protocol. Statistical analysis
The results are presented as mean – standard error and they were analyzed by SPSS16.0 software (SPSS). The differences between two groups were carried out by Student’s t-test. A p value of
DNA Methylation of DKK3 Modulates Docetaxel Chemoresistance in Human Nonsmall Cell Lung Cancer Cell Leilei Tao, Guichun Huang, Yitian Chen, and Longbang Chen
Abstract
Dickkopf-related protein 3 (DKK3) gene, as a tumor suppressor gene, has been discovered in various cancers, but its relationship with tumor chemoresistance is still unclear. In this study, this laboratory detected that DNA methylation contributes to the downregulation of DKK3 in docetaxel resistance of human lung cancer cells and its possible biochemical mechanism. DKK3 has been proved to be downregulated by hypermethylation in docetaxelresistant lung cancer cells. Upregulation of DKK3 can reverse the chemoresistance of docetaxel-resistant cell lines in vitro by growth inhibition and enhancement of apoptosis. Conversely, downregulation of DKK3 could induce parental human lung cancer cells insensitivity to docetaxel by promoting proliferative capacity and inhibiting apoptosis of cancer cells. In addition, the authors observed that overexpression of DKK3 might decrease the expression of P-glycoprotein. All results suggested that epigenetic downregulation of DKK3 leads to docetaxel resistance in human nonsmall cell lung cancer (NSCLC) cells by increased expression of P-glycoprotein. DKK3 may reveal a novel molecular target for docetaxel resistance for NSCLC patients in the future. Key words: chemoresistance, DKK3, DNA methylation, docetaxel, NSCLC Introduction
L
ung cancer is one of the highest incidence of malignancies and leading cause of cancer-related death in the world. It is reported that 80% of all lung cancer patients are nonsmall cell lung cancer (NSCLC). Most of the lung cancer patients are diagnosed in the advanced stages (Stages III and IV).1 Despite development of cancer treatment and introduction of new technology, advanced lung cancer remains associated with poor prognosis. Docetaxel, a semisynthetic analogue of paclitaxel, is widely used as an anticancer drug in the treatment of lung cancer.2 Docetaxel confers its antineoplastic activity by inhibiting microtubule depolymerization, leading the metaphase to anaphase transition arrest, activating the spindle assembly checkpoint, and subsequently causing apoptosis.3,4 However, chemotherapy agents become increasingly ineffective when the disease has spread beyond the lung at the advanced stage. Chemoresistance almost occurs in recurrent patients and remains the most important dilemma in restricting the clinical application of anticancer drugs.5 Thus, a further study of the molecular mechanism underlying
docetaxel resistance of NSCLC should be helpful to improve the efficacy of its clinical application. Accumulated evidences have proved that cancer cells resistant to docetaxel result from both genetic and epigenetic modification of various critical genes.6,7 Aberrant DNA methylation, leading to downregulation of key genes, which played a crucial role during the initiation and development of cancer.8 However, the correlation between DNA methylation and docetaxel resistance of NSCLC is rarely reported. In the laboratory, the authors previously established a docetaxel-resistant H1299 cell line (H1299/DTX) by continuous exposure to increasing concentrations of docetaxel. It is important for us to understand the molecular mechanisms underlying docetaxel resistance and explore novel therapeutic markers for reversing chemoresistance in NSCLC. Dickkopf-related protein 3 (DKK3), a putative Wnt antagonist, has been proved to represent as a tumor suppressor for downregulation in various human cancers. In many types of cancers, the failure of its normal expression is closely associated to CpG island methylation on the DKK3 promoter; thus, the published articles of DKK3 have mainly
Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University, Nanjing, People’s Republic of China. Address correspondence to: Longbang Chen; Department of Medical Oncology, Jinling Hospital, Medical School of Nanjing University; 305 ZhongShan Eastern Road, Nanjing 210002, People’s Republic of China. E-mail: [email protected]
100
DKK3 AND LUNG CANCER CHEMORESISTANCE
focused on the methylation-related field.9 The silencing expression of DKK3 is always correlated with poor prognosis of human cancer, including lung cancer,10 ovarian cancer,11 renal clear cell carcinoma,12 and prostate cancer.13 However, the association of DKK3 with chemotherapy resistance in NSCLC cells was seldom seen in the previous studies. It is vitally necessary that the molecular mechanism underlying docetaxel resistance should be elucidated and searched for novel therapeutic markers for reversing chemoresistance. In this study, the authors investigated that epigenetic downregulation of DKK3 mediated by aberrant promoter methylation contributes to docetaxel resistance in NSCLC cell lines. At the same time, DKK3 re-expression could reverse docetaxel resistance of lung cancer cell. The results of this study may provide a novel strategy for reversing chemoresistance of NSCLC. Materials and Methods Cell culture
Human NSCLC H1299 cells were bought from the Cell Bank of Chinese Academy of Medical Science. Cells were conditioned in the RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 lg/L streptomycin in a humidified atmosphere at 37C, 5% CO2. The docetaxel-resistant H1299 cell line (H1299/DTX) was established by continuous exposure to increasing concentrations of docetaxel, and cells were grown in the presence of 5 lg/L final concentration of docetaxel. RNA isolation and semiquantitative reverse transcription–polymerase chain reaction
Total RNA of tumor cells was isolated using the Trizol reagent (Invitrogen), and the spectrophotometer (BioPhotometer) was used to measure the concentration of total RNA. Reverse transcription (RT) was accomplished by the PrimeScript RT Reagent Kit (Takara) following the instruction manual of the manufacturer. Two micrograms of total RNA was applied for cDNA synthesis, and 20 lL reaction volume was prepared for further polymerase chain reaction (PCR). PCR conditions were in accordance with the manufacturer’s instructions. The primer sequences for each
101
gene were designed as shown in Table 1. The transcription of glyceraldehyde 3-phophate dehydrogenase (GAPDH) was performed for normalization. The PCR products were electrophoresed on 2% agarose gels and detected by ethidium bromide (EB) staining. Methylation-specific PCR
Methylation-specific PCR (MSP) was performed to detect the methylation status of DKK3 gene promoter by using bisulfate-modified genomic DNA as the template. Total DNA was isolated from cultured cells using the QIAamp DNA Mini Kit (Qiagen). One microgram of genomic DNA was bisulfate treated with the EZ-DNA methylation Gold Kit (Zymo Research) and finally resuspended in 10 lL TE buffer. Two sets of primers were used to display the differences between methylated and unmethylated status. The primers targeting the unmethylated and methylated DKK3 promoter regions are described in Table 1. The following steps were performed with an instruction manual. The PCR products were visualized in 2% agarose gel by EB staining. Lymphocyte DNA was used as unmethylation and methylation-positive control in the experiment. A water blank was performed as a negative control. Transient transfection plasmid or siRNA interference
The DKK3 expressing plasmid (pCS2-hDKK3) and negative control were available from Addgene (Plasmid 15496). Small interfering RNAs (siRNAs) targeting DKK3 (siRNA-DKK3) were obtained from Santa Cruz Biotechnology, Inc. Nonspecific control siRNA (siRNA/NC) was used as control. Plasmid DNA was extracted by the Plasmid Mini Kit (Qiagen). Cells were cultured in six-well plates (2 · 105cells/well) and transfected with the SiRNA Mate (GenePharma) or Turbofect Transfection reagent (Thermo Scientific) following the manufacturer’s protocol. Western blot
The cells were isolated directly or 72 hours after transfection; the cells were washed twice with phosphate buffered saline (PBS) before the proteins were extracted. Proteins were separated electrophoretically by 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene
Table 1. Primer Sequences Used for Quantitative PCR mRNA
Primers
DKK3
Forward: 5¢-GTTGAGGAACTGATGGAGGACA-3¢ Reverse: 5¢-TTGCACACATACACCAGGCTGT-3¢ Forward: 5¢-GGGGCGGGCGGCGGGGC-3¢ Reverse: 5¢-ACATCTCCGCTCTACGCCCG-3¢ Forward: 5¢-TTAGGGGTGGGTGGTGGGGT-3¢ Reverse: 5¢-CTACATCTCCACTCTACACCCA-3¢ Forward: 5¢-TGCGACAGGAGATAGGCTG-3¢ Reverse: 5¢-GCCAAAATCACAAGGGTTAGCTT-3¢ Forward: 5¢-GCACCGTCAAGGCTGAGAAC-3¢ Reverse: 5¢-TGGTGAAGACGCCAGTGGA-3¢
DKK3 (MSP-M) DKK3 (MSP-U) MDR-1 GAPDH
Expected size (bp) 695 118 126 193 138
PCR, polymerase chain reaction; DKK3, Dickkopf-related protein 3; MDR-1, multidrug resistance-1; GAPDH, glyceraldehyde 3phophate dehydrogenase.
102
fluoride (PVDF) membrane. The membranes were blocked for 2 hours with 5% nonfat milk at room temperature. The membranes were incubated first with the REIC/DKK3 antibody (1:2000; Abcam) or P-glycoprotein antibody (1:500; Bioss) overnight at 4C and then incubated with a second antibody for 2 hours at 37C. Proteins were then displayed with the ECL substrate (Cell Signaling Technology) in accordance with the manufacturer’s instructions. Protein levels were normalized to GAPDH. Cell viability assay
Single-cell suspensions were planted into 96-well plates (2 · 103 cells/well) directly or 24 hours after transfection. Various concentrations of docetaxel (0, 10, 20, 40, 80, 160, 320, 640, 1280 lg/L) were then added. Forty-eight hours later, cell viability was shown by the MTT assay. Colony formation assay
Three hundred cells were planted into each well in sixwell plates directly or 48 hours after transfection. After 14
TAO ET AL.
days, cells were fixed with methanol and stained with 0.1% crystal violet. Then, the number of visible colonies was counted. The cloning efficiency was calculated as follows: (the clone number/the number of plant cells) ·100%. Flow cytometric analysis of apoptosis
Cells were harvested 48 hours after transfection for cell apoptosis measurement, then resuspended in 0.5 mL binding buffer with 5 mL Annexin V and 5 mL propidium iodide (PI), and reacted for 15 minutes at 37C in the dark. Then, the early apoptotic rate was measured by flow cytometric analysis following the manufacturer’s protocol. Statistical analysis
The results are presented as mean – standard error and they were analyzed by SPSS16.0 software (SPSS). The differences between two groups were carried out by Student’s t-test. A p value of
