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Specific Amino Acid Profile in Culture Media Conditioned by Human Pancreatic Cancer Cell Lines
Original Paper
Pancratology
Pancreatology 2002;2:402-406
Received: April 30, 1999 Accepted: JLI1e 6, 2000
Specific Amino Acid Profile in Culture Media Conditioned by Human Pancreatic Cancer Cell Lines Feng Wang Johan Permert Department of Surgery, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden
KeyWords Pancreatic cancer· Amino acid profile· Culture media
Abstract Background: In malignant diseases, circulating amino acid profiles correlate with organ sites of malignancy. Direct effects of malignant cells on the extracellular amino acid profile are still uncertain. Methods: Free amino acids were measured in serum-free culture media (RPM 11640) conditioned by two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), a human epidermoid carcinoma cell line (A431), and a human fibroblastic cell line (Ag-1523). Nonconditioned RPMI-1640 medium was used as control. Results: Amino acid profiles were changed in all the conditioned media, caused by a decrease or increase in the original amino acids and by the appearance of amino acids that were not present in non-conditioned medium. Media conditioned by two human pancreatic cancer cell lines showed similar amino acid profiles, which were characterized by a decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine. Conclusion:Culture media show changed amino acid profiles following incubation with cell lines of pancreatic or non-pancreatic origins. Different human pancreatic cancer cell lines cause similar changes in amino acid profiles of media.
Introduction
Patients with malignant disease usually show abnormal amino acid profiles in the peripheral circulation [18]. Furthermore, changes in amino acid profile diagnostically correlate with organ sites of malignancy [1]. These observations indicate that malignant cells may have a direct influence on extracellular amino acid profiles. Exocrine pancreatic cancer is one of the leading causes of death from malignant disease. Direct effects of pancreatic cancer cells on the extracellular amino acid profile are still uncertain. In the present study, we investigated changes of amino acid contents in culture media conditioned by pancreatic cancer cells or by benign or malignant cells of non-pancreatic origins.
Materials and Methods Chemicals RPMI-1640, fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, and amphotericin-B were purchased from Life Technologies (Stockholm, Sweden). Cell lines investigated are shown in table 1. Panc-l and A-431 cells were bought from American Type Culture Collection (Rockville, Md., USA). Ag-1523 cells were purchased from Coriell Institute for Medical Research (Camden, N.J., USA). HPAF and PC-I cells were kindly donated by Dr. Parviz M. Pour (Omaha, N ebr., USA).
Copyright e 2002 S. Karger AG, Basel and lAP
KAR.GER.
© 2002 S, Karger AG, Basel and rAP 1424- 39 03/0 2/00 24-04 02$18 .5 0/0
Fax+41613051234 E-Mail [email protected] www.karger.com
Accessible online at
www.karger.com/journals/pan
Feng Wang, MD, PhD Surgery Laboratory, Clinical Research Centre, Novum Huddinge University Hospital S-14186 Huddinge (Sweden) Tel. +46 8 585 83865, Fax +46 8 585 83850, E-Mail feng,wang@karo,ki,se
Table 1. Cell lines investigated
Table 2. Glucose and amino acid concentrations in conditioned cul-
ture media Names
Donors
Sources
Panc-1
human
HPAF PC-I A-431 Ag-1523
human hamster human human
pancreatic adenocarcinoma (poorly differentiated) pancreatic adenocarcinoma (heterogeneous) pancreatic adenocarcinoma (ductal) epidennoid carcinoma fibroblastic cell (benign, passage No. 13)
Preparation of Conditioned Media Cell lines were suspended with RPMI-1640 containing 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ~glml streptomycin, and 2 ~glml amphotericin-B. The cell suspensions were seeded in culture flasks (75 cm 2) at densities of 2.8 x 10 4/cm 2 (Panc-1), 1.7 x 104/cm2 (HPAF), 1.1 x 104/cm2 (PC-I), 2.0 x 104/cm2 (A-43 I ) and 8.7 x 103/cm 2 (Ag-1523), respectively. The cell lines were cultured at 37°C in 95% humidified air and 5% CO2 , Culture media were changed every 48 h. When cells grew to 90% confluence, serum-containing media were removed. Cell cultures were rinsed three times with phosphate-buffered saline and incubated with 25 ml of serumfree RPMI -1640 media for 24 h. The conditioned media were harvested and centrifuged at 200 g for 10 min. Supernatants were collected and stored at - 20 0 C for subsequent assays. Cultured cells were trypsinized and cell viability was tested by trypan blue exclusion. Glucose and Amino Acid Concentrations in Conditioned Media Glucose concentrations in test media were measured by a reflectance photometer (Boehringer Mannheim, Mannheim, Gennany) using the glucose oxidase method. For amino acid analysis, incubation media were deproteinized with sulfosalicylic acid solution (35% w/v). Free amino acids were analysed by an automated amino acid analyser (Beckman System 6300, Beckman Instruments, Inc., Palo Alto, Calif., USA). The eluted amino acids were detennined by the ninhydrin-hydantoin reaction using S-2-aminoethyl-L-cysteine as internal standard [91. Statistics Statistic analyses were carried out using the computer program Instat (version 1.12, Graph Pad, San Diego, Calif., USA). Statistic significance was assessed using analysis of variance and the Bonferroni post-test for multiple comparisons. Values are mean ± SEM. A two-tailed p value of less than 0.05 was considered significant.
Results
Glucose and Total Amino Acids in Conditioned Media Glucose concentrations were decreased in culture media conditioned by four malignant cell lines (table 2). Total amino acid concentrations were significantly decreased in media conditioned by Panc-l and PC-l cells. In media conditioned by HPAF and A-431 cells, total
Pancreatic Cancer and Extracellular Amino Acids
Media
Glucose
mM
Total amino acids,mM
Bsential amino acids/total amino acids %
Control Panc-1 HPAF PC-I A-431 Ag-1523
10.6 ± 0.1 5.1 ±0.2*** 6.6 ± 0.2*** 7.9±0.1*** 8.0±0.1*** 10.2±0.1
6.3 ± 0.2 5.1 ±O.l* 5.5 ± 0.4 3.7±0.1*** 5.5±0.1 6.0±0.1
39.9 ± 0.3 43.1 ±0.1** 42.2 ± 0.2* 44.4 ± 0.8*** 42.7 ± 0.1 * 41.6 ± 0.3
Data are mean ± SEM, n = 4. * P < 0.05; 0.001.
** P < 0.01; *** P <
amino acid concentrations were slightly decreased but the changes did not reach statistical significance. Molar ratios of essential amino acids to total amino acids were increased in media conditioned by all the four malignant cell lines (table 2). Glucose and total amino acids in Ag1523 medium showed no significant differences compared to con troIs.
Changes in A mino Acid Profiles Amino acid profiles were altered in five conditioned media, compared with the non-conditioned control (fig. lA-E). Alterations were caused by a decrease or increase in original amino acids in RPMI-1640 media and by the appearance of non-original amino acids. Decrease in glutamine and cysteine and appearance of alanine were found in all the conditioned media (fig. lA-E). Media conditioned by benign fibroblastic cells only showed these common changes (fig. IE). Changes in other amino acids were seen in four media conditioned by different malignant cell lines (fig. lA-D). Media conditioned by two human pancreatic cancer cell lines (Panc-l and HPAF) showed similar alterations in amino acid profiles, including decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine (fig. lA, B). Some of the alterations, such as increase in glycine and proline and appearance of ornithine, were not found in other conditioned media. Concentrations of sixteen amino acids were significantly decreased in the media conditioned by PC-l cells (fig. lC). Among the four human cell lines, A-431 was the only cell line which decreased aspartic acid concentration in culture media (fig. lD).
Pancreatology 2002;2:402-406
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Discussion
Patients with malignant tumors usually show abnormal protein metabolism characterized by elevated wholebody protein turnover, enhanced hepatic protein synthesis, decreased muscle protein synthesis and increased muscle protein breakdown [10]. In addition, circulating amino acid profiles are changed in malignant tumor hosts [1-8]. In patients with cancers of the head and neck, breast, or gastrointestinal tract, circulating amino acid profiles diagnostically correlate with organ sites of malignancy [I]. In laboratory animals, removal of the tumor normalizes circulating amino acid profiles within 24 h [7]. These observations indicate that extracellular amino acid profiles may be affected by the amino acid metabolism of malignan t cells. In the present study, amino acid profiles of culture media were changed in different fashions by malignant cells of different origins. The results are in agreement with the concept that malignant cells have direct effects on the extracellular profile of amino acids. In this study, changed amino acid profiles in culture media were caused by a decrease and increase in original amino acids and by the appearance of non-original amino acids. Similarly, alterations of the circulating amino acid profile in tumor hosts are induced by both an increase and decrease in individual amino acids [1-8]. Furthermore, molar ratios between
Fig. 1. Serum-free culture media were incubated for 24 h with two
human pancreatic cancer cell lines, Panc-l (A) and HPAF (B), a hamster pancreatic cancer cellline PC-l (C), a human epidennoid carcinoma cell1ine A-431 (D) and a human fibroblastic cell line Ag1523 (E). Amino acid contents in the media were measured and results were expressed as alteration of amino acids compared with non-conditioned medium. Data are mean ± SEM, n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001. 0 Amino acids not present in non-conditioned medium. gln = Glutamine; cys = cysteine; ser = serine; asn = asparagine; leu = leucine; ile = isoleucine; thr = threonine; phe = phenylalanine; asp = aspartic acid; arg = arginine; hyp = hydroxyproline; lys = lysine; tyr = tyrosine; val = valine; his = histidine; met = methionine; trp = trypotophan; om = ornithine; ala = alanine; gly = glycine; pro = proline; glu = glutamic acid.
Pancreatic Cancer and Extracellular Amino Acids
essential amino acids and total amino acids were increased in the media prepared from four malignant cell lines but not in the media from benign fibroblastic cells. In vivo, the ratio between essential and total amino acids is increased in the peripheral circulation of patients with malignant tumors [I]. In the culture media conditioned by four malignant cells, amino acid profiles were altered, to a great extent, by a decrease in individual amino acid concentrations. As a result, total amino acid concentrations were significantly decreased in Pane-I and PC-I media and insignificantly decreased in HPAF and A-431 media. Besides, glucose concentrations in these media were significantly decreased. These results indicate that malignant cells have high rates of nutrient utilization. In contrast, increased amino acid concentrations and appearance of non-original amino acids in conditioned media may result from a release of these amino acids from the cultured cells. It is notable that media conditioned by two human pancreatic cancer cell lines (Pane-I and HPAF) showed similar changes in media amino acid profiles. Some of the changes, such as an increase in glycine and proline and appearance of ornithine, were not found in the other media. The changed amino acid profiles in Pane-I and HPAF media potentially reflected characteristics of amino acid metabolism in human pancreatic cancer cells. Kubota et al. [I] demonstrated that seven amino acids, i.e. glutamine, threonine, histidine, cysteine, alanine, arginine and ornithine, correlate highly with diagnoses of tumor sites. In the present study, four of these seven amino acids were altered in both Pane-I and HPAF media. The normal exocrine pancreas has a high rate of amino acid metabolism, as has been approved in vivo by Domschke et al. [II, 12]. Frazier et al. [13] found that gene products of the normal exocrine pancreas, such as RNAs for carbonic anhydrase and trypsin, were also expressed in malignant exocrine pancreatic cells, suggesting that the malignant cells retain some characteristics of protein production of the normal exocrine pancreas. Thus, the amino acid metabolism in pancreatic cancer cells may cause special extracellular amino acid profiles in vitro. For future studies, it is necessary to characterize circulating amino acids in patients with pancreatic cancer. Such a study can tell us whether the in vitro effects of human pancreatic cancer cells on extracellular amino acids still exist in vivo. A specific circulating amino acid profile in patients with pancreatic cancer will be instrumental for early diagnosis of pancreatic cancer. In addition, human pancreatic cancer cells are usually of ductal
Pancreatology 2002;2:402-406
405
origin. A cell line has been established from human pancreatic ducts [14]. It is tempting to compare amino acid profiles of culture media conditioned either by human pancreatic cancer cells or by benign pancreatic ductal cells.
Acknowledgment This work was supported by grants from the Swedish Cancer Society (2870-B95-05XBB and 2870-B96-06XAC).
References
2
3
4
5
Kubota A, Meguid MM, Hitch DC: Amino acid profiles correlate diagnostically with organ site in three kinds of malignant tumours. Cancer 1992;69:2343-2348. Cascina A, Muscaritoli M, Cangiano C, Conversano L, Laviano A, Ariemma S, Meguid MM, Fanelli FR: Plasma amino acid imbalance in patients with lung and breast cancer. Anticancer Res 1995;15:507-510. Ching N, Grossi C, Jham G, Angers J, Zurawinsky H , Ching CY, Nealon TF: Plasma amino acid and serum unesterified fatty acid deficits and the effect of nutritional support in chemotherapy treatment. Surgery 1984;95:730737. Norton JA, Gorschboth eM, Wesley RA, Burt ME, Brennan MF: Fasting plasma amino acid levels in cancer patients. Cancer 1985;56: 1181-1186. Kelley JJ, Waisman HA: Quantitative plasma amino acid values in leukemic blood. Blood 1957; 12635-643.
406
6 Landel AM, Lo CC, Meguid MM, Rivera D: Effect of methylcholanthrene-induced sarcoma and its removal on rat plasma and intracellular free amino acid content. Surg Res Comm 1987; 1273-287. 7 Chance WT, Cao L, Fischer JE: Inducedinduced alterations in brain neurotransmitter and plasma ammonia concentrations are normalized twenty-four hours after tumour resection. Life &i 1991;48:425-432. 8 Watanabe A, Higashi T, Sakata T, Nagashima H: Serum amino acid levels in patients with hepatocellular carcinoma. Cancer 1984;54: 1875-1882. 9 Yanhanen I, Svedjeholm R, Hakanson E, Joachimsson PO, Jorfeldt L, Nilsson L, Yanky F: Assessment of myocardial glutamate requirements early after coronary artery bypass surgery. &and CardiovascJ 1998;32:145-152. 10 Rossi Fanelli F, Cangiano C, Muscaritoli M, Conversano L, Torelli GF, Cascino A: Induced-induced changes in host metabolism: a possible marker of neoplastic disease. Nutrition 1995;11:595-600.
Pancreatology 2002;2:402-406
11 Domschke S, Heptner G, Kolb S, Sailer D, Schneider MU, Domschke W: Deacreas in plasma amino acid level after secretin and pancreozymin as an indicator of exocrine pancreatic function. Gastroenterology 1986;90:10311038. 12 Fisher H, Konturek JW, Szlachcic A, Konturek SJ, Domschke: Plasma amino acids consumption and pancreatic secretion during and after cerulein-induced pancreatitis in rats. Int J PancreatoI1995;18:127-134. 13 Frazier ML, Fernandez E, de Llorens R, Brown NM, Pathak S, Cleary KR, AbbruzzeseJL, Berry K, Olive M, Le Maistre A, Evans DB: Pancreatic adenocarcinoma cell line, MDAPanc28, with features of both acinar and ductal cells. IntJ PancreatoI1996;19:31-38. 14 Trautmann B, &hlitt HJ, Hahn EG, L6hr M: Isolation, culture, and characterization of human pancreatic duct cells. Pancreas 1993;8: 248-254.
WangiPennert
Pancratology
Pancreatology 2002;2:402-406
Received: April 30, 1999 Accepted: JLI1e 6, 2000
Specific Amino Acid Profile in Culture Media Conditioned by Human Pancreatic Cancer Cell Lines Feng Wang Johan Permert Department of Surgery, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden
KeyWords Pancreatic cancer· Amino acid profile· Culture media
Abstract Background: In malignant diseases, circulating amino acid profiles correlate with organ sites of malignancy. Direct effects of malignant cells on the extracellular amino acid profile are still uncertain. Methods: Free amino acids were measured in serum-free culture media (RPM 11640) conditioned by two human pancreatic cancer cell lines (Panc-1 and HPAF), a hamster pancreatic cancer cell line (PC-1), a human epidermoid carcinoma cell line (A431), and a human fibroblastic cell line (Ag-1523). Nonconditioned RPMI-1640 medium was used as control. Results: Amino acid profiles were changed in all the conditioned media, caused by a decrease or increase in the original amino acids and by the appearance of amino acids that were not present in non-conditioned medium. Media conditioned by two human pancreatic cancer cell lines showed similar amino acid profiles, which were characterized by a decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine. Conclusion:Culture media show changed amino acid profiles following incubation with cell lines of pancreatic or non-pancreatic origins. Different human pancreatic cancer cell lines cause similar changes in amino acid profiles of media.
Introduction
Patients with malignant disease usually show abnormal amino acid profiles in the peripheral circulation [18]. Furthermore, changes in amino acid profile diagnostically correlate with organ sites of malignancy [1]. These observations indicate that malignant cells may have a direct influence on extracellular amino acid profiles. Exocrine pancreatic cancer is one of the leading causes of death from malignant disease. Direct effects of pancreatic cancer cells on the extracellular amino acid profile are still uncertain. In the present study, we investigated changes of amino acid contents in culture media conditioned by pancreatic cancer cells or by benign or malignant cells of non-pancreatic origins.
Materials and Methods Chemicals RPMI-1640, fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, and amphotericin-B were purchased from Life Technologies (Stockholm, Sweden). Cell lines investigated are shown in table 1. Panc-l and A-431 cells were bought from American Type Culture Collection (Rockville, Md., USA). Ag-1523 cells were purchased from Coriell Institute for Medical Research (Camden, N.J., USA). HPAF and PC-I cells were kindly donated by Dr. Parviz M. Pour (Omaha, N ebr., USA).
Copyright e 2002 S. Karger AG, Basel and lAP
KAR.GER.
© 2002 S, Karger AG, Basel and rAP 1424- 39 03/0 2/00 24-04 02$18 .5 0/0
Fax+41613051234 E-Mail [email protected] www.karger.com
Accessible online at
www.karger.com/journals/pan
Feng Wang, MD, PhD Surgery Laboratory, Clinical Research Centre, Novum Huddinge University Hospital S-14186 Huddinge (Sweden) Tel. +46 8 585 83865, Fax +46 8 585 83850, E-Mail feng,wang@karo,ki,se
Table 1. Cell lines investigated
Table 2. Glucose and amino acid concentrations in conditioned cul-
ture media Names
Donors
Sources
Panc-1
human
HPAF PC-I A-431 Ag-1523
human hamster human human
pancreatic adenocarcinoma (poorly differentiated) pancreatic adenocarcinoma (heterogeneous) pancreatic adenocarcinoma (ductal) epidennoid carcinoma fibroblastic cell (benign, passage No. 13)
Preparation of Conditioned Media Cell lines were suspended with RPMI-1640 containing 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ~glml streptomycin, and 2 ~glml amphotericin-B. The cell suspensions were seeded in culture flasks (75 cm 2) at densities of 2.8 x 10 4/cm 2 (Panc-1), 1.7 x 104/cm2 (HPAF), 1.1 x 104/cm2 (PC-I), 2.0 x 104/cm2 (A-43 I ) and 8.7 x 103/cm 2 (Ag-1523), respectively. The cell lines were cultured at 37°C in 95% humidified air and 5% CO2 , Culture media were changed every 48 h. When cells grew to 90% confluence, serum-containing media were removed. Cell cultures were rinsed three times with phosphate-buffered saline and incubated with 25 ml of serumfree RPMI -1640 media for 24 h. The conditioned media were harvested and centrifuged at 200 g for 10 min. Supernatants were collected and stored at - 20 0 C for subsequent assays. Cultured cells were trypsinized and cell viability was tested by trypan blue exclusion. Glucose and Amino Acid Concentrations in Conditioned Media Glucose concentrations in test media were measured by a reflectance photometer (Boehringer Mannheim, Mannheim, Gennany) using the glucose oxidase method. For amino acid analysis, incubation media were deproteinized with sulfosalicylic acid solution (35% w/v). Free amino acids were analysed by an automated amino acid analyser (Beckman System 6300, Beckman Instruments, Inc., Palo Alto, Calif., USA). The eluted amino acids were detennined by the ninhydrin-hydantoin reaction using S-2-aminoethyl-L-cysteine as internal standard [91. Statistics Statistic analyses were carried out using the computer program Instat (version 1.12, Graph Pad, San Diego, Calif., USA). Statistic significance was assessed using analysis of variance and the Bonferroni post-test for multiple comparisons. Values are mean ± SEM. A two-tailed p value of less than 0.05 was considered significant.
Results
Glucose and Total Amino Acids in Conditioned Media Glucose concentrations were decreased in culture media conditioned by four malignant cell lines (table 2). Total amino acid concentrations were significantly decreased in media conditioned by Panc-l and PC-l cells. In media conditioned by HPAF and A-431 cells, total
Pancreatic Cancer and Extracellular Amino Acids
Media
Glucose
mM
Total amino acids,mM
Bsential amino acids/total amino acids %
Control Panc-1 HPAF PC-I A-431 Ag-1523
10.6 ± 0.1 5.1 ±0.2*** 6.6 ± 0.2*** 7.9±0.1*** 8.0±0.1*** 10.2±0.1
6.3 ± 0.2 5.1 ±O.l* 5.5 ± 0.4 3.7±0.1*** 5.5±0.1 6.0±0.1
39.9 ± 0.3 43.1 ±0.1** 42.2 ± 0.2* 44.4 ± 0.8*** 42.7 ± 0.1 * 41.6 ± 0.3
Data are mean ± SEM, n = 4. * P < 0.05; 0.001.
** P < 0.01; *** P <
amino acid concentrations were slightly decreased but the changes did not reach statistical significance. Molar ratios of essential amino acids to total amino acids were increased in media conditioned by all the four malignant cell lines (table 2). Glucose and total amino acids in Ag1523 medium showed no significant differences compared to con troIs.
Changes in A mino Acid Profiles Amino acid profiles were altered in five conditioned media, compared with the non-conditioned control (fig. lA-E). Alterations were caused by a decrease or increase in original amino acids in RPMI-1640 media and by the appearance of non-original amino acids. Decrease in glutamine and cysteine and appearance of alanine were found in all the conditioned media (fig. lA-E). Media conditioned by benign fibroblastic cells only showed these common changes (fig. IE). Changes in other amino acids were seen in four media conditioned by different malignant cell lines (fig. lA-D). Media conditioned by two human pancreatic cancer cell lines (Panc-l and HPAF) showed similar alterations in amino acid profiles, including decrease in glutamine, cysteine and serine, increase in glycine, proline and glutamic acid and appearance of ornithine and alanine (fig. lA, B). Some of the alterations, such as increase in glycine and proline and appearance of ornithine, were not found in other conditioned media. Concentrations of sixteen amino acids were significantly decreased in the media conditioned by PC-l cells (fig. lC). Among the four human cell lines, A-431 was the only cell line which decreased aspartic acid concentration in culture media (fig. lD).
Pancreatology 2002;2:402-406
403
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Discussion
Patients with malignant tumors usually show abnormal protein metabolism characterized by elevated wholebody protein turnover, enhanced hepatic protein synthesis, decreased muscle protein synthesis and increased muscle protein breakdown [10]. In addition, circulating amino acid profiles are changed in malignant tumor hosts [1-8]. In patients with cancers of the head and neck, breast, or gastrointestinal tract, circulating amino acid profiles diagnostically correlate with organ sites of malignancy [I]. In laboratory animals, removal of the tumor normalizes circulating amino acid profiles within 24 h [7]. These observations indicate that extracellular amino acid profiles may be affected by the amino acid metabolism of malignan t cells. In the present study, amino acid profiles of culture media were changed in different fashions by malignant cells of different origins. The results are in agreement with the concept that malignant cells have direct effects on the extracellular profile of amino acids. In this study, changed amino acid profiles in culture media were caused by a decrease and increase in original amino acids and by the appearance of non-original amino acids. Similarly, alterations of the circulating amino acid profile in tumor hosts are induced by both an increase and decrease in individual amino acids [1-8]. Furthermore, molar ratios between
Fig. 1. Serum-free culture media were incubated for 24 h with two
human pancreatic cancer cell lines, Panc-l (A) and HPAF (B), a hamster pancreatic cancer cellline PC-l (C), a human epidennoid carcinoma cell1ine A-431 (D) and a human fibroblastic cell line Ag1523 (E). Amino acid contents in the media were measured and results were expressed as alteration of amino acids compared with non-conditioned medium. Data are mean ± SEM, n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001. 0 Amino acids not present in non-conditioned medium. gln = Glutamine; cys = cysteine; ser = serine; asn = asparagine; leu = leucine; ile = isoleucine; thr = threonine; phe = phenylalanine; asp = aspartic acid; arg = arginine; hyp = hydroxyproline; lys = lysine; tyr = tyrosine; val = valine; his = histidine; met = methionine; trp = trypotophan; om = ornithine; ala = alanine; gly = glycine; pro = proline; glu = glutamic acid.
Pancreatic Cancer and Extracellular Amino Acids
essential amino acids and total amino acids were increased in the media prepared from four malignant cell lines but not in the media from benign fibroblastic cells. In vivo, the ratio between essential and total amino acids is increased in the peripheral circulation of patients with malignant tumors [I]. In the culture media conditioned by four malignant cells, amino acid profiles were altered, to a great extent, by a decrease in individual amino acid concentrations. As a result, total amino acid concentrations were significantly decreased in Pane-I and PC-I media and insignificantly decreased in HPAF and A-431 media. Besides, glucose concentrations in these media were significantly decreased. These results indicate that malignant cells have high rates of nutrient utilization. In contrast, increased amino acid concentrations and appearance of non-original amino acids in conditioned media may result from a release of these amino acids from the cultured cells. It is notable that media conditioned by two human pancreatic cancer cell lines (Pane-I and HPAF) showed similar changes in media amino acid profiles. Some of the changes, such as an increase in glycine and proline and appearance of ornithine, were not found in the other media. The changed amino acid profiles in Pane-I and HPAF media potentially reflected characteristics of amino acid metabolism in human pancreatic cancer cells. Kubota et al. [I] demonstrated that seven amino acids, i.e. glutamine, threonine, histidine, cysteine, alanine, arginine and ornithine, correlate highly with diagnoses of tumor sites. In the present study, four of these seven amino acids were altered in both Pane-I and HPAF media. The normal exocrine pancreas has a high rate of amino acid metabolism, as has been approved in vivo by Domschke et al. [II, 12]. Frazier et al. [13] found that gene products of the normal exocrine pancreas, such as RNAs for carbonic anhydrase and trypsin, were also expressed in malignant exocrine pancreatic cells, suggesting that the malignant cells retain some characteristics of protein production of the normal exocrine pancreas. Thus, the amino acid metabolism in pancreatic cancer cells may cause special extracellular amino acid profiles in vitro. For future studies, it is necessary to characterize circulating amino acids in patients with pancreatic cancer. Such a study can tell us whether the in vitro effects of human pancreatic cancer cells on extracellular amino acids still exist in vivo. A specific circulating amino acid profile in patients with pancreatic cancer will be instrumental for early diagnosis of pancreatic cancer. In addition, human pancreatic cancer cells are usually of ductal
Pancreatology 2002;2:402-406
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origin. A cell line has been established from human pancreatic ducts [14]. It is tempting to compare amino acid profiles of culture media conditioned either by human pancreatic cancer cells or by benign pancreatic ductal cells.
Acknowledgment This work was supported by grants from the Swedish Cancer Society (2870-B95-05XBB and 2870-B96-06XAC).
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