Apoptosis Induced by Glucose-6-phosphate Dehydrogenase Deficiency is Mediated by Caspase-3 and PARP-1 in Human Melanoma Cells

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1 ISSN CN / Q Chinese Journal of Biochemistry and Molecular Biology ~ 1127 G6PD Caspase-3 PARP * G6PD HEM A375 A375-WT G6PD A375 A375-G6PDΔ real-time PCR Western A375-WT mrna G6PD G6PD HEM 1 89 P < P < P < 0 05 Annexin V / PI Western A375- G6PDΔ A375-WT 5 10 P < caspase P < kd -1 PARP P < 0 01 A375-G6PDΔ NADPH GSH 72 30% P < % P < % H 2 P < 0 01 G6PD G6PD caspase-3 PARP caspase-3-1 PARP-1 R Q554 Apoptosis Induced by Glucose-6-phosphate Dehydrogenase Deficiency is Mediated by Caspase-3 and PARP-1 in Human Melanoma Cells HAO Xiao-Pei 1 REN Na 1 1 TANG Qiong-Lin 2 ZHANG Chun-Hua 1 HU Tao 1 CHEN Long 1 ZHU Yue-Chun 1 * 1 Department of Biochemistry Kunming Medical University Kunming China 2 Shenzhen Louhu Maternal and Child Health Hospital Shenzhen China Abstract The mechanism of overexpression of glucose-6-phosphate dehydrogenase G6PD in many tumor cells is no still largely understood To elucidate the relationship between G6PD and human melanoma human epidermal melanocyte HEM human melanoma A375 A375-WT and G6PD deficient A375 A375-G6PDΔ were used to study the development of cell apoptosis and its mechanism Real-time PCR and Western blotting showed that mrna and protein of G6PD in A375-WT cells increased by 1 89 times P < 0 05 and 6 86 times P < 0 01 respectively with 2 30 times P < No No 2007PY01-13 * Tel yuechunzhu@ gmail com Received September Accepted October Supported by National Natural Science Foundation of China No and Yunnan Provincial Science and Technology Foundation No 2007PY * Corresponding author Tel yuechunzhu@ gmail com These authors contributed equally to this work

2 increase of G6PD activity as those of HEM cells Further study showed that a apoptotic rate increased by 5 10-fold P < 0 01 in A375-G6PDΔ cells with annexin V / PI flow cytometry analysis and cleaved caspase-3 and 89 kd PARP-1 raised by 1 84-fold 2 87-fold respectively In addition A375-G6PDΔ cells showed that levels of NADPH and GSH decreased by 72 30% P < % P < 0 05 respectively with 75 43% increase of H 2 P < 0 01 as those of A375-WT cells These results suggest that the apoptosis induced by G6PD deficiency may be mediated by caspase-3 and PARP-1 in human melanoma cells which may be a new clue in the study of melanoma tumorigenesis Key words glucose-6-phosphate dehydrogenase melanoma caspase-3 PARP-1 cell apoptosis malignant melanoma 1 100U / ml 100U / ml DMEM 37 5% C G6PD A375 A375-G6PDΔ G6PD GenBank NM_ sirna A375-WT G6PD 200 μg / ml 3 caspase-3 G % 0 02% EDTA aspase- human epidermal melanocytes HEM caspase-3 1 ~ 2 3 ~ 4 6 5% C 37 60% -1 poly ADP-ribose polymerase-1 PARP-1 caspase-3 4% 30 min Kim 5 G361 1 PBS 3 0 1% L- L-Dopa PARP-1 caspase h 1 L-Dopa Ⅱ NADPH ROS reactive oxygen 3 species 1 2 Annexin V / PI NADPH EDTA 0 25% -6- glucose-6-phosphate dehydrogenase G6PD EC μl annexin V 5 μl PI AV / PI GSH 6 G6PD A375 Backman EPICS XL-4CIR 488 nm 525 nm annexin V 620 nm PI annexin V G6PD PI caspase-3 PARP-1 annexin V PI 1 10% 7 A375-G6PDΔ G418 ~ 70% 1 PBS 2 ~ / ml r / min 5 min PBS μl binding buffer 2 KGA108 Nanking 20 min PBS 2 annexin V PI 3 / 1 3 G6PD caspase-3 PARP-1 RNA GAPDH 1 1 PCR Sybrgreen G6PD A375-WT mrna G6PD-F 5 -TGAGCCAGA

3 12 G6PD Caspase-3 PARP TAGGCTGGAA-3 G6PD-R 5 -TAACGCAGGCGATGTT GTC-3 GAPDH-F 5 -AGCCTCAAGATCATCAGC-3 GAPDH-R 5 -GAGTCCTTCCACGATACC-3 G6PD GAPDH 96 bp 225 bp G6PD caspase-3 pro caspase-3 cleaved PARP kd PARP-1 89 kd 2 1 G6PD A375 HEM β-actin HRP HEM HRP L- dopa β-actin Western G6PD Fig 1A caspase-3 PARP-1 L-dopa HEM SensiAnsys 1 4 G6PD NADPH GSH H 2 25 HITACHI U nm GFP NADP + Sigma NADPH 2 2 G6PD A375 G6PD 6 min mg 1μmol / L NADPH 1 IU 10 3 Braford 1 5 x 珋 ± S t P < 99% A375-G6PDΔ GFP sirna prna-u6 2 / Lenti Fig 1C 99% 20 NEM A375-WT mrna G6PD G6PD 1 89 ± 0 42 NADPH GSH 0 24 ± ± P Catalog # A061 H 2 Catalog # A061 < 0 05 Fig 2 A 6 86 P < 0 01 Fig 2 B C 2 30 P < 0 05 Fig 2 D G6PD Fig 2 The expressions of G6PD and its activities in the NEM A375-WT and A375-G6PDΔ cells A The mrna level of G6PD was checked with real-time PCR Total RNA was isolated from NEM A375-WT and A375-G6PDΔ cells cdna of G6PD gene was synthesized by RT-PCR G6PD mrna levels were normalized as the ratio of the fluorescence intensity from G6PD to that of GAPDH P < 0 05 A375-WT vs NEM and P < 0 01 A375-G6PDΔ vs A375-WT B The analysis of G6PD expression with Western blotting Cell lysates were prepared The proteins in lysates were separated by 10% SDS- PAGE After transferring into the membrane the blots were probed with anti-human G6PD antibody β-actin was used as a loading control C The relative amount of G6PD expression was obtained by the image analysis software SensiAnsys from Western blotting P < 0 01 both A375-WT vs NEM and A375-G6PDΔ vs A375-WT D The assay of G6PD activities by the ultraviolet spectrometry G6PD activity was measured by its ability to reduce 2 mm of NADP + in the presence of glucose 6- phosphate P < 0 05 A375-WT vs NEM and P < 0 01 A375-G6PDΔ vs A375-WT The photographs showed here were representative of three experiments

4 Fig 1 Identifications of HEM cells and A375-G6PDΔ cells A HEM cells were obtained by primary culture from children prepuce and fixed with 4% formaldehyde solution then were stained 3 hours with 0 1% L-dopa solution and taken a picture under an optical microscope with 10 magnification B A375- G6PDΔ cells under an optical microscope with 10 magnification C All A375-G6PDΔ cells were GFP positive under a fluorescence microscope with 10 magnification which were a stable line of A375 human melanoma cells with G6PD gene knockdown by a shrna lentiviral cloning and expression system A375-WT annexin V / PI G6PD A375-G6PDΔ G6PD A ± 7 24 A375-WT 9 14 ± 1 83 A375-G6PDΔ G6PD mrna G6PD A375 A375-WT 22 22% 24 17% 28 49% P < G6PD A P < 0 01 Fig 3A B G6PD 2 4 G6PD Caspase-3 PARP-1 G6PD A375 caspase-3 PARP-1 A375-G6PDΔ Western Fig 4A B A375-G6PDΔ caspase-3 32 kd A375-WT 6 G6PD procaspase-3 17 kd cleaved Fig 3 Cell apoptosis analysis with annexin V / PI staining and flow cytometry A Cells were harvested and washed in cold PBS Added 2μL annexinv and 5 μl PI AV / PI Apoptosis Detection Kit to each 100 μl of cell suspension then incubated the cells at room temperature for 20 minutes and analyzed the stained cells by flow cytometry The apoptotic cells showed positive with annexin V staining B The apoptosis rate of cell was calculated based on the cells with annexin V staining P < 0 01 A375-G6PDΔ vs A375-WT The photographs showed here were representative of three experiments

5 12 G6PD Caspase-3 PARP caspase ± 0 19 A375-WT 0 86 ± 0 15 A375-WT 1 72 ± 0 53 P < ± 0 09 P < A375-G6PDΔ cleaved G6PD caspase-3 caspase kd PARP-1 89 kd PARP-1 Fig 4 The analysis of caspase-3 acting on PARP-1 with Western blotting A The analysis of caspase-3 activity and incision on 116 kd PARP-1 with Western blotting Cell lysates were prepared The proteins in lysates were separated by 10% SDS-PAGE After transferring into the membrane the blots were probed with anti-human procaspase-3 cleaved caspase kd PARP-1 and 89 kd PARP-1 antibody β-actin was used as a loading control B The relative amount of procaspase-3 cleaved caspase kd PARP-1 and 89 kd PARP-1 expression were obtained by the image analysis software SensiAnsys from Western blotting The results showed that the protein expression levels of both cleaved caspase-3 and 89 kd PARP-1 in A375-G6PDΔ were greatly higher than those of A375-WT P < 0 01 The photographs showed here were representative of three experiments Table 1 Productions of NADPH GSH and H 2 in A375-G6PDΔ and A375-WT 珔 x ± s NADPH GSH H 2 U / mg % U / mg % U / mg % A375-WT ± ± ± A375-G6PDΔ ± ± ± vs 1 P < vs 4 P < G6PD A375 GSH H 2 Table 1 Fig 5 A375- G6PD WT A375-G6PDΔ NADPH NADPH GSH 72 30% P < % P < 0 05 H 2 O 2 NADPH 75 43% P < ~ 15 Murtas 3 P53 re- G6PD programming G6PD 16 G6PD G6PD A375 NEM Fig 2 sirna A375-WT G6PD P < 0 01 Fig 3A G6PD 3B Procaspase kd PARP-1 89 kd Fig 4A 4B PARP-1 G6PD G6PD

6 caspase-3 PARP-1 H 2 procaspase-3 Fig 6 Fig 5 Levels of NADPH GSH and H 2 with spectrophotometric method The cells were harvested and subjected to a freeze-thaw to lyse The cell lysates containing the cytosolic components were then used to measure the contents of NADPH GSH and H 2 with a spectrophotometer according to the manufacturer's instruction Jiancheng Nanjing China All measurements were repeated three times with the blank controls filled with media but without cells The results showed that levels of both NADPH and GSH decreased with an increase of H 2 in the A375-G6PDΔ P < 0 05 G6PD A375 procaspase-3 an important factor in the pathway but the mechanism in PARP-1 H 2 activation caspase-3 is still largely unknown and need PARP-1 to be further investigated RNA DNA DNA References DNA DNA 17 Nguewa 18 PARP-1 DNA DNA PARP kd PARP-1 ADP- DNA DNA G6PD A375 H 2 GSH Lin 19 H 2 DNA procaspase-3 caspase-3 PARP-1 DNA caspase-3 89 kd 20 6 Li D Y Zhu Y Tang Q L et al H 2 susceptibility to oxidative stress J caspase NF-кB c DNA 21 H 2 O 2 G6PD A375 Fig 6 The pathway analysis of A375 cell apoptosis induced by G6PD deficiency Based on the results as described in Fig 3 4 and 5 the pathway of A375 cells apoptosis induced by G6PD deficiency was showed H 2 is 1 Lens M B Dawes M Global perspectives of contemporary epidemiological trends of cutaneous malignant melanoma J Br J Dermatol Garbe C Eigentler T K Diagnosis and treatment of cutaneous melanoma state of the art 2006 J Melanoma Res Pop C Salvesen G S Human caspases activation specificity and regulation J J Biol Chem Denault J B Salvesen G S Apoptotic caspase activation and activity J Methods Mol Biol Kim D S Jeon S E Jeong Y M et al Hydrogen peroxide is a mediator of indole-3-acetic acid / horseradish peroxidase-induced apoptosis J FEBS Lett A new G6PD knockdown tumor-cell line with reduced proliferation and increased Cancer Biother Radiopharm 7 sirna G6PD J Zhu Yue-Chun Lv Hui-Ru Li Dan-Yi et al Effects of silencing G6PD expression on the growth and apoptosis in human

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