Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97725
標題: 研究新穎的DNA甲基轉移酶抑制劑對人類胰臟癌細胞抗癌效用及分子機轉
The study of anticancer effects and molecular mechanisms by a novel DNA methyltransferase inhibitor on human pancreatic cancer cells.
作者: 黃茂軒
Mao-Hsuan Huang
關鍵字: 胰臟癌
正丁烯基苯酞
DNA甲基轉移酶1
patched domain containing 4
刺蝟訊息傳遞路徑
Pancreatic ductal adenocarcinoma
n-Butylidenephthalide
DNA-methyltransferase 1
patched domain containing 4
Hedgehog pathway
引用: 1. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. The New England journal of medicine. 2014;371:2140-2141 2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for clinicians. 2016;66:7-30 3. Porta M, Fabregat X, Malats N, Guarner L, Carrato A, de Miguel A, Ruiz L, Jariod M, Costafreda S, Coll S, Alguacil J, Corominas JM, Sola R, Salas A, Real FX. Exocrine pancreatic cancer: Symptoms at presentation and their relation to tumour site and stage. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico. 2005;7:189-197 4. Harsha HC, Kandasamy K, Ranganathan P, Rani S, Ramabadran S, Gollapudi S, Balakrishnan L, Dwivedi SB, Telikicherla D, Selvan LD, Goel R, Mathivanan S, Marimuthu A, Kashyap M, Vizza RF, Mayer RJ, Decaprio JA, Srivastava S, Hanash SM, Hruban RH, Pandey A. A compendium of potential biomarkers of pancreatic cancer. PLoS medicine. 2009;6:e1000046 5. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA: a cancer journal for clinicians. 2018;68:7-30 6. Bosetti C, Lucenteforte E, Silverman DT, Petersen G, Bracci PM, Ji BT, Negri E, Li D, Risch HA, Olson SH, Gallinger S, Miller AB, Bueno-de-Mesquita HB, Talamini R, Polesel J, Ghadirian P, Baghurst PA, Zatonski W, Fontham E, Bamlet WR, Holly EA, Bertuccio P, Gao YT, Hassan M, Yu H, Kurtz RC, Cotterchio M, Su J, Maisonneuve P, Duell EJ, Boffetta P, La Vecchia C. Cigarette smoking and pancreatic cancer: An analysis from the international pancreatic cancer case-control consortium (panc4). Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2012;23:1880-1888 7. Shi C, Hruban RH, Klein AP. Familial pancreatic cancer. Archives of pathology & laboratory medicine. 2009;133:365-374 8. Hassan MM, Bondy ML, Wolff RA, Abbruzzese JL, Vauthey JN, Pisters PW, Evans DB, Khan R, Chou TH, Lenzi R, Jiao L, Li D. Risk factors for pancreatic cancer: Case-control study. The American journal of gastroenterology. 2007;102:2696-2707 9. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. The New England journal of medicine. 2014;371:1039-1049 10. Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M, Hruban RH, Maitra A, Kinzler K, Vogelstein B, Goggins M. Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology. 2012;142:730-733 e739 11. Hustinx SR, Leoni LM, Yeo CJ, Brown PN, Goggins M, Kern SE, Hruban RH, Maitra A. Concordant loss of mtap and p16/cdkn2a expression in pancreatic intraepithelial neoplasia: Evidence of homozygous deletion in a noninvasive precursor lesion. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc. 2005;18:959-963 12. Korc M. Pancreatic cancer-associated stroma production. American journal of surgery. 2007;194:S84-86 13. Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, Hingorani SR. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer cell. 2012;21:418-429 14. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu CC, Simpson TR, Laklai H, Sugimoto H, Kahlert C, Novitskiy SV, De Jesus-Acosta A, Sharma P, Heidari P, Mahmood U, Chin L, Moses HL, Weaver VM, Maitra A, Allison JP, LeBleu VS, Kalluri R. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer cell. 2014;25:719-734 15. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM. Identification of pancreatic cancer stem cells. Cancer research. 2007;67:1030-1037 16. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, Bruns CJ, Heeschen C. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell stem cell. 2007;1:313-323 17. Martin RC, 2nd, Scoggins CR, Egnatashvili V, Staley CA, McMasters KM, Kooby DA. Arterial and venous resection for pancreatic adenocarcinoma: Operative and long-term outcomes. Archives of surgery. 2009;144:154-159 18. Further evidence of effective adjuvant combined radiation and chemotherapy following curative resection of pancreatic cancer. Gastrointestinal tumor study group. Cancer. 1987;59:2006-2010 19. Burris HA, 3rd, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: A randomized trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 1997;15:2403-2413 20. de Sousa Cavalcante L, Monteiro G. Gemcitabine: Metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. European journal of pharmacology. 2014;741:8-16 21. Moore MJ, Goldstein D, Hamm J, Figer A, Hecht JR, Gallinger S, Au HJ, Murawa P, Walde D, Wolff RA, Campos D, Lim R, Ding K, Clark G, Voskoglou-Nomikos T, Ptasynski M, Parulekar W, National Cancer Institute of Canada Clinical Trials G. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: A phase iii trial of the national cancer institute of canada clinical trials group. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2007;25:1960-1966 22. Wang-Gillam A, Li CP, Bodoky G, Dean A, Shan YS, Jameson G, Macarulla T, Lee KH, Cunningham D, Blanc JF, Hubner RA, Chiu CF, Schwartsmann G, Siveke JT, Braiteh F, Moyo V, Belanger B, Dhindsa N, Bayever E, Von Hoff DD, Chen LT, Group N-S. Nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine-based therapy (napoli-1): A global, randomised, open-label, phase 3 trial. Lancet. 2016;387:545-557 23. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693-705 24. Robertson KD, Jones PA. DNA methylation: Past, present and future directions. Carcinogenesis. 2000;21:461-467 25. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, Wolffe AP. Methylated DNA and mecp2 recruit histone deacetylase to repress transcription. Nature genetics. 1998;19:187-191 26. Robertson KD. DNA methylation and human disease. Nature reviews. Genetics. 2005;6:597-610 27. Esteller M. Epigenetics in cancer. The New England journal of medicine. 2008;358:1148-1159 28. Subramaniam D, Thombre R, Dhar A, Anant S. DNA methyltransferases: A novel target for prevention and therapy. Frontiers in oncology. 2014;4:80 29. Du Q, Wang Z, Schramm VL. Human dnmt1 transition state structure. Proceedings of the National Academy of Sciences of the United States of America. 2016;113:2916-2921 30. Etoh T, Kanai Y, Ushijima S, Nakagawa T, Nakanishi Y, Sasako M, Kitano S, Hirohashi S. Increased DNA methyltransferase 1 (dnmt1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple cpg islands in gastric cancers. The American journal of pathology. 2004;164:689-699 31. Li A, Omura N, Hong SM, Goggins M. Pancreatic cancer dnmt1 expression and sensitivity to dnmt1 inhibitors. Cancer biology & therapy. 2010;9:321-329 32. Nagpal G, Sharma M, Kumar S, Chaudhary K, Gupta S, Gautam A, Raghava GP. Pcmdb: Pancreatic cancer methylation database. Scientific reports. 2014;4:4197 33. Kanda T, Tada M, Imazeki F, Yokosuka O, Nagao K, Saisho H. 5-aza-2'-deoxycytidine sensitizes hepatoma and pancreatic cancer cell lines. Oncology reports. 2005;14:975-979 34. Gros C, Fahy J, Halby L, Dufau I, Erdmann A, Gregoire JM, Ausseil F, Vispe S, Arimondo PB. DNA methylation inhibitors in cancer: Recent and future approaches. Biochimie. 2012;94:2280-2296 35. Yang CS, Wang X, Lu G, Picinich SC. Cancer prevention by tea: Animal studies, molecular mechanisms and human relevance. Nature reviews. Cancer. 2009;9:429-439 36. Lin PC, Chen YL, Chiu SC, Yu YL, Chen SP, Chien MH, Chen KY, Chang WL, Lin SZ, Chiou TW, Harn HJ. Orphan nuclear receptor, nurr-77 was a possible target gene of butylidenephthalide chemotherapy on glioblastoma multiform brain tumor. Journal of neurochemistry. 2008;106:1017-1026 37. Yen SY, Chen SR, Hsieh J, Li YS, Chuang SE, Chuang HM, Huang MH, Lin SZ, Harn HJ, Chiou TW. Biodegradable interstitial release polymer loading a novel small molecule targeting axl receptor tyrosine kinase and reducing brain tumour migration and invasion. Oncogene. 2016;35:2156-2165 38. Huang MH, Lin SZ, Lin PC, Chiou TW, Harn YW, Ho LI, Chan TM, Chou CW, Chuang CH, Su HL, Harn HJ. Brain tumor senescence might be mediated by downregulation of s-phase kinase-associated protein 2 via butylidenephthalide leading to decreased cell viability. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35:4875-4884 39. Jiang J, Hui CC. Hedgehog signaling in development and cancer. Developmental cell. 2008;15:801-812 40. Briscoe J, Therond PP. The mechanisms of hedgehog signalling and its roles in development and disease. Nature reviews. Molecular cell biology. 2013;14:416-429 41. Oliver TG, Grasfeder LL, Carroll AL, Kaiser C, Gillingham CL, Lin SM, Wickramasinghe R, Scott MP, Wechsler-Reya RJ. Transcriptional profiling of the sonic hedgehog response: A critical role for n-myc in proliferation of neuronal precursors. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:7331-7336 42. Pasca di Magliano M, Sekine S, Ermilov A, Ferris J, Dlugosz AA, Hebrok M. Hedgehog/ras interactions regulate early stages of pancreatic cancer. Genes Dev. 2006;20:3161-3173 43. Onishi H, Katano M. Hedgehog signaling pathway as a new therapeutic target in pancreatic cancer. World journal of gastroenterology : WJG. 2014;20:2335-2342 44. Harn HJ, Lin SZ, Lin PC, Liu CY, Liu PY, Chang LF, Yen SY, Hsieh DK, Liu FC, Tai DF, Chiou TW. Local interstitial delivery of z-butylidenephthalide by polymer wafers against malignant human gliomas. Neuro-oncology. 2011;13:635-648 45. Hidalgo M. Pancreatic cancer. The New England journal of medicine. 2010;362:1605-1617 46. Feng M, Xiong G, Cao Z, Yang G, Zheng S, Song X, You L, Zheng L, Zhang T, Zhao Y. Pd-1/pd-l1 and immunotherapy for pancreatic cancer. Cancer letters. 2017;407:57-65 47. Vincent A, Omura N, Hong SM, Jaffe A, Eshleman J, Goggins M. Genome-wide analysis of promoter methylation associated with gene expression profile in pancreatic adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2011;17:4341-4354 48. Gao J, Wang L, Xu J, Zheng J, Man X, Wu H, Jin J, Wang K, Xiao H, Li S, Li Z. Aberrant DNA methyltransferase expression in pancreatic ductal adenocarcinoma development and progression. Journal of experimental & clinical cancer research : CR. 2013;32:86 49. Wang W, Gao J, Man XH, Li ZS, Gong YF. Significance of DNA methyltransferase-1 and histone deacetylase-1 in pancreatic cancer. Oncology reports. 2009;21:1439-1447 50. Zhang JJ, Zhu Y, Zhu Y, Wu JL, Liang WB, Zhu R, Xu ZK, Du Q, Miao Y. Association of increased DNA methyltransferase expression with carcinogenesis and poor prognosis in pancreatic ductal adenocarcinoma. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico. 2012;14:116-124 51. Xu M, Gao J, Du YQ, Gao DJ, Zhang YQ, Li ZS, Zhang YL, Gong YF, Xu P. Reduction of pancreatic cancer cell viability and induction of apoptosis mediated by sirna targeting dnmt1 through suppression of total DNA methyltransferase activity. Molecular medicine reports. 2010;3:699-704 52. Missiaglia E, Donadelli M, Palmieri M, Crnogorac-Jurcevic T, Scarpa A, Lemoine NR. Growth delay of human pancreatic cancer cells by methylase inhibitor 5-aza-2'-deoxycytidine treatment is associated with activation of the interferon signalling pathway. Oncogene. 2005;24:199-211 53. Fryer RA, Barlett B, Galustian C, Dalgleish AG. Mechanisms underlying gemcitabine resistance in pancreatic cancer and sensitisation by the imid lenalidomide. Anticancer research. 2011;31:3747-3756 54. Voutsadakis IA. Molecular predictors of gemcitabine response in pancreatic cancer. World journal of gastrointestinal oncology. 2011;3:153-164 55. Datta J, Ghoshal K, Denny WA, Gamage SA, Brooke DG, Phiasivongsa P, Redkar S, Jacob ST. A new class of quinoline-based DNA hypomethylating agents reactivates tumor suppressor genes by blocking DNA methyltransferase 1 activity and inducing its degradation. Cancer research. 2009;69:4277-4285 56. Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, Lauwers GY, Qi YP, Gysin S, Fernandez-del Castillo C, Yajnik V, Antoniu B, McMahon M, Warshaw AL, Hebrok M. Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature. 2003;425:851-856 57. Chung JH, Larsen AR, Chen E, Bunz F. A ptch1 homolog transcriptionally activated by p53 suppresses hedgehog signaling. The Journal of biological chemistry. 2014;289:33020-33031 58. He S, Wang F, Yang L, Guo C, Wan R, Ke A, Xu L, Hu G, Xu X, Shen J, Wang X. Expression of dnmt1 and dnmt3a are regulated by gli1 in human pancreatic cancer. PloS one. 2011;6:e27684 59. Sekiya K, Tezuka Y, Tanaka K, Prasain JK, Namba T, Katayama K, Koizumi T, Maeda M, Kondo T, Kadota S. Distribution, metabolism and excretion of butylidenephthalide of ligustici chuanxiong rhizoma in hairless mouse after dermal application. Journal of ethnopharmacology. 2000;71:401-409 60. Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, Lin SZ, Harn HJ. The natural compound n-butylidenephthalide derived from angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. Journal of neurochemistry. 2006;99:1251-1262 61. Neesse A, Michl P, Frese KK, Feig C, Cook N, Jacobetz MA, Lolkema MP, Buchholz M, Olive KP, Gress TM, Tuveson DA. Stromal biology and therapy in pancreatic cancer. Gut. 2011;60:861-868 62. Feig C, Gopinathan A, Neesse A, Chan DS, Cook N, Tuveson DA. The pancreas cancer microenvironment. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18:4266-4276 63. Leach KJ, Mathiowitz E. Degradation of double-walled polymer microspheres of plla and p(cpp:Sa)20:80. I. In vitro degradation. Biomaterials. 1998;19:1973-1980 64. Sipos EP, Tyler B, Piantadosi S, Burger PC, Brem H. Optimizing interstitial delivery of bcnu from controlled release polymers for the treatment of brain tumors. Cancer chemotherapy and pharmacology. 1997;39:383-389 65. Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, Zheng S, Chakravarty D, Sanborn JZ, Berman SH, Beroukhim R, Bernard B, Wu CJ, Genovese G, Shmulevich I, Barnholtz-Sloan J, Zou L, Vegesna R, Shukla SA, Ciriello G, Yung WK, Zhang W, Sougnez C, Mikkelsen T, Aldape K, Bigner DD, Van Meir EG, Prados M, Sloan A, Black KL, Eschbacher J, Finocchiaro G, Friedman W, Andrews DW, Guha A, Iacocca M, O'Neill BP, Foltz G, Myers J, Weisenberger DJ, Penny R, Kucherlapati R, Perou CM, Hayes DN, Gibbs R, Marra M, Mills GB, Lander E, Spellman P, Wilson R, Sander C, Weinstein J, Meyerson M, Gabriel S, Laird PW, Haussler D, Getz G, Chin L, Network TR. The somatic genomic landscape of glioblastoma. Cell. 2013;155:462-477 66. Collisson EA, Sadanandam A, Olson P, Gibb WJ, Truitt M, Gu S, Cooc J, Weinkle J, Kim GE, Jakkula L, Feiler HS, Ko AH, Olshen AB, Danenberg KL, Tempero MA, Spellman PT, Hanahan D, Gray JW. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 2011;17:500-503 67. Indolfi L, Ligorio M, Ting DT, Xega K, Tzafriri AR, Bersani F, Aceto N, Thapar V, Fuchs BC, Deshpande V, Baker AB, Ferrone CR, Haber DA, Langer R, Clark JW, Edelman ER. A tunable delivery platform to provide local chemotherapy for pancreatic ductal adenocarcinoma. Biomaterials. 2016;93:71-82 68. Yeo CJ, Cameron JL, Lillemoe KD, Sitzmann JV, Hruban RH, Goodman SN, Dooley WC, Coleman J, Pitt HA. Pancreaticoduodenectomy for cancer of the head of the pancreas. 201 patients. Annals of surgery. 1995;221:721-731; discussion 731-723 69. Konstantinidis IT, Warshaw AL, Allen JN, Blaszkowsky LS, Castillo CF, Deshpande V, Hong TS, Kwak EL, Lauwers GY, Ryan DP, Wargo JA, Lillemoe KD, Ferrone CR. Pancreatic ductal adenocarcinoma: Is there a survival difference for r1 resections versus locally advanced unresectable tumors? What is a 'true' r0 resection? Annals of surgery. 2013;257:731-736
摘要: 胰臟癌在美國是因癌症死亡人數的第四位,患有胰臟癌的病人只有8% 可以活到五年以上,是一種極為致命的疾病。主要原因是目前臨床用來治療胰臟癌的化療藥物,其抗癌效果皆不佳,最多只延長三個月的壽命。異常的DNA甲基化在胰臟的癌化過程中被認為扮演重要的角色,同時將近50%的胰臟癌病人DNA甲基轉移酶1 (DNMT1) 都有過度表達的現象。因此我們以DNMT1的表現當作篩選平台,從中找到一個新的DNMTs抑制劑,正丁烯基苯酞 (n-butylidenephthalide, n-BP),n-BP可以劑量依賴性和時間依賴性方式抑制DNMT1的表現。同時我們也發現n-BP治療人類胰臟癌細胞後,可以有效的抑制胰臟癌細胞生長。以流式細胞儀分析它對細胞週期的影響,結果顯示n-BP不只會造成細胞週期停滯於G0/G1,也會引發細胞凋亡的現象。在用基因轉殖技術維持DNMT1的表現後,n-BP造成的細胞抑制生長現象就被大幅降低。此外我們也發現n-BP是透過影響蛋白質穩定性去抑制DNMT1而不是透過RNA的轉錄。為了更進一步了解n-BP降低DNMT1後影響了哪些基因,我們利用微陣列分析技術掃描發現,patched domain containing 4 (PTCHD4) 是DNMT1的潛在下游基因。在透過siRNA沉默PTCHD4表現後,n-BP便無法降低腫瘤的生長。而且PTCHD4 是一個腫瘤抑制基因參與在Hedgehog路徑上,我們也看到n-BP能夠誘導PTCHD4進而抑制Hedgehog路徑的活化。更進一步,我們利用兩種動物模式來評估n-BP在活體內的療效。透過局部緩慢釋放高分子聚合物來攜帶n-BP進行治療,實驗的結果不論是皮下腫瘤或原位瘤的動物模式,BP都能有效的抑制胰臟癌的生長,同時藉由免疫組織切片染色,我們觀察到與細胞實驗一致的結果。總合以上結果證實,n-BP可能是透過降低DNMT1並增加PTCHD4的表現來抑制人類胰臟癌細胞的生長,同時在體外和體內都有很好的抗癌效果,顯示n-BP有潛力做為未來治療胰臟癌的標靶藥物。
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with only eight percent of the patients surviving beyond five years. One of the major reasons is the low effectiveness of anticancer drugs that only extends survival by 3 months. Aberrant DNA methylation has been considered to play an important role during carcinogenesis in PDAC, with approximately 50% of tumor tissues overexpressing the DNA methyltransferase 1 (DNMT1) protein. In the present study, we used DNMTs as a screening platform to find a new DNMT inhibitor, n-butylidenephthalide (n-BP). n-BP could inhibit DNMT1 expression in both dose-dependent and time-dependent manner. It also displays an effect in suppressing growth of PDAC cells, inducing cell cycle arrest at G0/G1 phase, and apoptosis. Growth suppression can be restored by the overexpression of DNMT1 in PDAC cells. Furthermore, we found n-BP-mediated DNMT1 suppression influenced the protein stability rather than changing the RNA expression. Through microarray analysis, we found that the patched domain contained 4 (PTCHD4) is the potential downstream gene of DNMT1. Following silencing of PTCHD4 expression by siRNA, n-BP decreased tumor growth inhibition. PTCHD4 is also a tumor suppressor gene involved in the Hedgehog pathway. We observed that n-BP could induce PTCHD4 and inhibit the activation of the Hedgehog pathway. Finally, in vivo, two animal models were used to evaluate the efficacy and survival after n-BP treatment by interstitial control release polymer delivery. The results show that n-BP could effectively inhibit PDAC tumor volume growth and extend animal survival. In summary, n-BP may inhibit the growth of human PDAC cells though reducing DNMT1 and increasing the expression of PTCHD4 both in vitro and in vivo.
URI: http://hdl.handle.net/11455/97725
文章公開時間: 2021-01-11
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.