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NADH Oxidase Activity from Sera Altered by Capsaicin Is Widely Distributed among Cancer Patients
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 342, No. 2, June 15, pp. 224–230, 1997 Article No. BB970110
NADH Oxidase Activity from Sera Altered by Capsaicin Is Widely Distributed among Cancer Patients D. James Morre´,*,1 Sara Caldwell,* Adrienne Mayorga,* Lian-Ying Wu,† and Dorothy M. Morre´† Departments of *Medicinal Chemistry and Molecular Pharmacology and †Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907
Received December 10, 1996, and in revised form March 19, 1997
A cancer-specific form of NADH oxidase inhibited or stimulated by 1 or 100 mM capsaicin (8-methyl-Nvanillyl-6-noneamide) is present in sera from cancer patients. The capsaicin-inhibited NADH oxidase activity appears to be absent from sera of individuals free of cancer. The capsaicin-inhibited activity is present both in freshly collected sera and in sera stored frozen for varying periods of time. For the latter, an assay was carried out under renaturing conditions in the presence of NADH and reduced glutathione followed by dilute hydrogen peroxide. Inhibition was half maximal at about 1 mM capsaicin. The capsaicin-inhibited activity was found in sera over a broad spectrum of cancer patients including patients with solid cancers (e.g., breast, prostate, lung, ovarian) as well as with leukemias and lymphomas. q 1997 Academic Press Key Words: cancer; serum; NADH oxidase; capsaicin.
Our laboratory described a capsaicin-inhibited NADH oxidase activity associated with plasma membranes of cancer cells grown in culture (1). The EC50 for inhibition of plasma membrane NADH oxidase activity and growth in BT-20 mammary carcinoma cells was about 1 mM (1). A similar capsaicin-inhibited activity was subsequently shown to appear in conditioned culture media of HeLa cells (2) as a shed form of reduced molecular weight. This observation prompted a study to determine if a similar activity might be shed into sera by autochthonous tumors in patients. MATERIALS AND METHODS Sera. Patient sera were from the patient population of the Michiana Hematology–Oncology Clinic (South Bend, IN) and from the 1
To whom correspondence should be addressed at Department of Medicinal Chemistry and Molecular Pharmacology, HANS Life Sciences Research Building, Purdue University, West Lafayette, IN 47907. Fax: (765) 494-4007.
patient population of Arnett Clinic (Lafayette, IN). Sera from normal volunteers were collected by the Michiana Hematology–Oncology Clinic and the Purdue University Health Center. Informed consent was obtained and confidentiality of medical records was assured by assigning a number to each serum sample. Patients were identified only as with active disease (e.g., stage III or stage IV) at the time of collection and as to organ site. Capsaicin (8-methyl-N-vanillyl-6-noneamide) was from Sigma (St. Louis, MO) and dissolved in DMSO.2 The final concentration of DMSO in the assay was 0.1% for 1 mM capsaicin and 0.2% for 100 mM capsaicin. Blanks without capsaicin contained DMSO. Spectrophotometric NADH oxidase assay. NADH oxidase activity was determined at 377C as the disappearance of NADH measured at 340 nm. Activity was measured using a Hitachi U3210 spectrophotometer with stirring and continuous recording over 5-min intervals. The reaction mixture contained 25 mM Tris–Mes buffer (pH 7.2), 1 mM KCN, 150 mM NADH, and 100 ml serum. A millimolar extinction coefficient of 6.22 was used to calculate the rate of NADH disappearance. Frozen samples were assayed by the addition to the complete reaction mixture of 2.5 ml 100 mM reduced glutathione (100 mM final concentration). After 5 to 15 min incubation at 377C, the assay was started by the addition of 2.5 ml 30% hydrogen peroxide (0.03% final concentration) and 2.5 ml 1 mM ATP (final concentration 1 mM). The activity was assayed over 10 to 15 min until a stable steady state was achieved after which 2.5 ml 1 mM capsaicin in DMSO was added (1 mM final concentration). The assay was continued for 10 min, after which 2.5 ml of 100 mM capsaicin in DMSO was added (final concentration of 100 mM) and the assay continued for an additional 10 min. An equivalent amount of DMSO alone was without effect.
RESULTS
Human sera from normal individuals contained NADH oxidase activity (Fig. 1). The activity was sensitive to heating (10 min at 807C). It was proportional to serum concentration and time of incubation (Fig. 1A). The response to NADH was biphasic. The Km for NADH was determined to be 25 mM over the range of 0 to 50 mM NADH and ca. 125 mM over the range 90 to 350 mM NADH (Fig. 1B). 2 Abbreviations used: DMSO, dimethyl sulfoxide; Mes, 4-morpholineethanesulfonic acid; PCMB, p-chloromercuribenzoic acid.
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FIG. 1. Enzymatic characteristics of NADH oxidation by human sera. (A) Proportionality to serum amount and time (inset). (B) Response to NADH concentration.
The activity from sera of cancer patients was stimulated by GTP (Fig. 2) and inhibited by GDP (Fig. 3). The dose–response for pooled sera from cancer patients and from normal volunteers was very similar. The activity was inhibited, as well, by the thiol reagents PCMB (Fig. 4) and N-ethylmaleimide (not shown). The oxidation of NADH was more sensitive to PCMB for cancer sera than for normal sera. The EC50 for sera from cancer patients was 10 nM PCMB, whereas that for sera of healthy volunteers was about ca. 0.1 mM PCMB. A differential response to capsaicin was observed with sera pooled from cancer patients (Fig. 5). NADH oxidase activity was inhibited half maximally by 100 mM capsaicin and maximally by about 1 mM capsaicin. NADH activity of sera pooled from healthy volunteers was unaffected by capsaicin over the same range of capsaicin concentrations that inhibited that of the sera of cancer patients. To begin to investigate the generality of the capsaicin inhibition of NADH oxidase activity of sera from cancer patients, individually collected sera were examined. Values are presented in the order that the data were collected. No values were excluded except for two samples where no rates were obtained. The majority of the samples had been collected within 6 months of assay and were stored frozen. With apparently healthy volunteers, the rate of NADH oxidation varied over a range of 10-fold from
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FIG. 2. Stimulation of NADH oxidase activity of human sera by GTP. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
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FIG. 3. Inhibition of NADH oxidase activity of human sera by GDP. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
FIG. 5. Response to capsaicin of the NADH oxidase activity of pooled sera from cancer patients (open symbols) and from healthy volunteers (solid symbols). Results are averages from three different lots of sera { SD.
FIG. 4. Inhibition of NADH oxidase activity of human sera by PCMB. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
0.09 to 0.9 nmol/min/100 ml serum (Table I). The source of this variation is unknown but may relate to the time of holding at room temperature prior to freezing. Activity declined progressively over several hours at room temperature. However, with none of the sera from healthy volunteers did capsaicin affect the activity by more than 5%. Overall, with sera from healthy volunteers, both 1 mM capsaicin and 100 mM capsaicin were without effect. With solid tumors, values were less variable and with three exceptions (breast cancer sample 5 that was stimulated by addition of 1 mM capsaicin and a myeloma sample and a leukemia sample (sample 2) that were stimulated by addition of 100 mM capsaicin), all were inhibited in activity by both 1 and 100 mM capsaicin in DMSO (Table II). Activity was, on average, greater with sera from cancer patients (0.7 nmol/min/100 ml serum) than for healthy volunteers (0.46 nmol/min/100 ml serum). With sera of cancer patients, inhibition was about 22% on average with 1 mM capsaicin in DMSO and about 38% on average with 100 mM capsaicin in DMSO. Inhibition at 100 mM capsaicin was greatest with colon cancer (53%) and least with prostate cancer (26%). The mean inhibited rate of NADH oxidation in the presence of 100 mM capsaicin of 0.4 nmol/min/100 ml serum for sera of cancer patients compared closely to that of sera of apparently healthy laboratory volunteers in the absence of capsaicin (0.46 nmol/min/100 ml serum).
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NADH OXIDASE OF CANCER SERA TABLE I
NADH Oxidase Activities of Sera from Apparently Healthy Laboratory Volunteers and Response to 1 and 100 mM Capsaicin in DMSO Capsaicin addition
Sample
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
1 2 3 4 5 6 7 8 9 10 Mean
0.09 0.11 0.56 0.56 0.12 0.6 0.14 0.92 0.92 0.6 0.46
0.09 0.11 0.56 0.58 0.12 0.58 0.14 0.92 0.96 0.64 0.47
0.00 0.00 0.00 0.02 0.00 00.02 0.00 0.00 0.04 0.04 0.01 { 0.02
1.0 1.0 1.0 1.04 1.0 0.97 1.0 1.0 1.04 1.07 1.01
0.09 0.11 0.58 0.56 0.13 0.58 0.14 0.88 0.92 0.64 0.46
0.00 0.00 0.02 0.00 0.01 00.02 0.00 00.04 0.00 0.04 0.00 { 0.02
1.0 1.0 1.04 1.0 1.08 0.97 1.0 0.96 1.0 1.07 1.01
1.0 1.0 1.02 1.02 1.04 0.97 1.0 0.98 1.02 1.07 1.01
Note. All data in nmol/min/100 ml sera.
With leukemias and lymphomas (Table III), results were similar to those for solid tumors. Serum NADH oxidase averaged 0.7 nmol/min/100 ml serum and was 30% inhibited by 1 mM capsaicin in DMSO and 45% inhibited by 100 mM capsaicin in DMSO. The solid cancers all were represented by metastatic disease, e.g., stage III or IV, and the organ site designations refer to the primary cancer. There was no selection to indicate any general proximate causes such as immunosuppression. We have checked interference by differences in age, sex, and particular medications and none were indicated. Sera collected in parallel to the sera from cancer patients were available for a limited number of disorders other than cancer. Results are summarized in Table IV. Although mean values for NADH oxidase activity were, on average, higher than for normal, no response to capsaicin outside of the normal range was observed with any of the sera. DISCUSSION
Our laboratory previously reported a hormone- and growth factor-stimulated NADH oxidase activity of the mammalian plasma membrane (3). The activity was present as well in plasma membranes of rat hepatomas but was no longer growth factor and hormone responsive (4). The activity in plasma membranes from rat liver was stimulated by GTP and other nucleoside triphosphates and inhibited by GDP (5). The activity from hepatoma and HeLa cell membranes, but not that from rat liver, was inhibited by thiol reagents including PCMB and N-ethylmaleimide (6). A further characteristic of the NADH oxidase activity of plasma mem-
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branes from HeLa and other transformed cell lines was its ability to be inhibited by capsaicin (8-methyl-N-vanillyl-6-noneamide) (1). The ability of capsaicin to inhibit NADH oxidase activity correlated with the ability of NADH oxidase to induce apoptosis and prevent the growth of tumor cells (1). Capsaicin inhibited NADH oxidase activity and growth of human cervical carcinoma (HeLa), adenocarcinoma, ovarian carcinoma, HL-60, K-562, and melanoma cells but not of mammary epithelia or melanocytes (1, 7). Also not inhibited was NADH oxidation by plasma membrane vesicles from rat liver, normal rat kidney cells, and Chinese hamster ovary cells. A capsaicin-inhibited NADH oxidase activity similar to that observed with plasma membranes isolated from HeLa cells was found to be present in culture media conditioned by the growth of HeLa cells (2). This activity was identified, based on antisera raised against the protein from culture media, to represent a shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane. The shed form of the activity showed the same response to capsaicin and to thiol reagents as the plasma membraneassociated form. These findings led us to seek evidence for a shed form of the capsaicin-inhibited NADH oxidase in sera of patients with active cancer. Human sera did contain NADH oxidase activity. The Km for NADH and response to guanine nucleotides was similar to that of the plasma membrane-associated form (3, 5). The NADH oxidase activity of serum from cancer patients with active disease also exhibited a greater sensitivity to the thiol reagent PCMB than was observed with sera of
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TABLE II
NADH Oxidase Activities of Sera from Cancer Patients with Active Disease and Response to 1 and 100 mM Capsaicin in DMSO Capsaicin addition
Cancer Breast 1 2 3 4 5 6 7 8 9 10 Mean Prostate 1 2 3 4 5 6 7 8 9 10 Mean Colon 1 2 3 4 5 Mean Lung 1 2 3 4 5 Mean Other Ovarian Myeloma Esophageal Ovarian Melanoma Mean Note. All data in * Significant P ** Significant P *** Significant P † Significant P †† Significant P ††† Significant P ‡ Significant P ‡‡ Significant P
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None (1)
1 mM (2)
Ratio 2/1
100 mM (3)
0.38 0.84 0.72 1.12 0.32 0.72 1.00 0.68 0.60 0.32 0.67
0.26 0.68 0.50 0.96 0.48 0.64 0.28 0.56 0.46 0.16 0.50
1.20 1.00 0.68 1.04 1.28 1.16 0.54 0.40 1.08 1.28 0.97
Ratio 3/1
Average ratio
00.12 00.16 00.22 00.16 0.16 00.08 00.72 00.12 00.14 00.16 00.17 { 0.18*
0.68 0.81 0.69 0.86 1.5 0.89 0.28 0.82 0.77 0.50 0.78
0.28 0.52 0.48 0.16 0.20 0.44 0.60 0.56 0.36 0.14 0.37
00.10 00.32 00.24 00.96 00.12 00.28 00.40 00.12 00.24 00.18 00.30 { 0.25**
0.74 0.62 0.67 0.14 0.63 0.61 0.60 0.82 0.60 0.44 0.59
0.71 0.71 0.68 0.5 (1.07) 0.75 0.44 0.82 0.69 0.47 0.69
0.88 0.80 0.56 0.88 1.16 0.96 0.44 0.32 0.96 1.04 0.80
00.32 00.20 00.12 00.16 00.12 00.20 00.10 00.08 00.12 00.24 00.17 { 0.7†
0.73 0.8 0.82 0.85 0.91 0.83 0.81 0.8 0.89 0.81 0.83
0.68 0.76 0.52 0.88 0.92 0.68 0.48 0.36 0.68 0.96 0.69
00.52 00.24 00.16 00.16 00.36 00.48 00.06 00.04 00.40 00.96 00.28 { 0.17†
0.57 0.76 0.76 0.85 0.72 0.59 0.89 0.9 0.63 0.75 0.74
0.65 0.78 0.79 0.85 0.81 0.71 0.85 0.85 0.76 0.78 0.78
0.46 0.22 0.20 1.12 0.48 0.50
0.22 0.06 0.04 0.92 0.38 0.32
00.24 00.18 00.16 00.20 00.10 00.18 { 0.05††
0.48 0.27 0.2 0.82 0.79 0.51
0.16 0.00 0.18 0.32 0.38 0.21
00.30 00.22 00.02 00.80 00.10 00.29 { 0.31†††
0.35 0 0.9 0.29 0.79 0.47
0.41 0.13 0.55 0.55 0.79 0.49
0.72 0.68 0.72 0.68 0.56 0.67
0.64 0.48 0.68 0.52 0.50 0.56
00.08 00.20 00.04 00.16 00.06 00.11 { 0.07***
0.89 0.71 0.94 0.76 0.89 0.84
0.56 0.18 0.56 0.56 0.44 0.46
00.16 00.50 00.16 00.12 00.12 00.21 { 0.16††
0.78 0.26 0.78 0.82 0.79 0.69
0.83 0.49 0.86 0.79 0.84 0.76
1.08 0.16 1.20 0.46 0.56 0.69
0.92 0.12 0.72 0.32 0.48 0.51
00.16 00.04 00.48 00.14 00.08 00.18 { 0.17‡
0.85 0.75 0.6 0.69 0.86 0.75
0.96 0.24 0.28 0.28 0.40 0.43
00.12 0.08 00.92 00.18 00.16 00.26 { 0.35‡‡
0.89 1.5 0.23 0.61 0.71 0.79
0.87 (1.13) 0.41 0.65 0.79 0.77
D
nmol/min/100 ml sera. õ 0.0056. õ 0.00014. õ 0.0008. õ 0.0001. õ 0.001. õ 0.0074. õ 0.0031. õ 0.03.
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D
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NADH OXIDASE OF CANCER SERA TABLE III
NADH Oxidase Activity of Sera from Patients with Active Leukemia or Lymphomas and Response to 1 or 100 mM Capsaicin in DMSO Capsaicin addition
Disease Leukemia 1 Granulocytic 2 Granulocytic 3 Granulocytic 4 Lymphocytic 5 Lymphocytic Mean Lymphoma (non-Hodgkin’s) 1 2 3 4 5 Mean
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
0.18 0.30 1.12 0.88 0.64 0.63
0.12 0.20 0.84 0.72 0.40 0.46
00.06 00.10 00.28 00.16 00.24 00.17 { 0.09*
0.67 0.67 0.75 0.82 0.63 0.71
0.00 0.32 0.72 0.68 0.36 0.42
00.18 0.02 00.40 00.20 00.28 00.21 { 0.14**
0 1.07 0.64 0.77 0.56 0.61
0.33 (0.87) 0.69 0.79 0.59 0.65
0.48 1.04 0.68 1.08 0.66 0.79
0.26 0.72 0.52 0.68 0.56 0.55
00.22 00.32 00.16 00.40 00.10 00.24 { 0.12*
0.54 0.69 0.76 0.63 0.85 0.69
0.06 0.52 0.52 0.44 0.44 0.40
00.42 00.52 00.16 00.64 00.22 00.39 { 0.20*
0.13 0.5 0.76 0.41 0.67 0.49
0.33 0.59 0.76 0.52 0.76 0.59
Note. All data in nmol/min/100 ml sera. * Significant P õ 0.0001. ** Significant P õ 0.0003.
apparently healthy volunteers. A similar phenomenon was observed with the NADH oxidase activity of HeLa plasma membranes compared to that of rat liver. The shed form of the activity found in sera of cancer patients also was stimulated by GTP and inhibited by GDP. A similar response was seen previously with the NADH oxidase of rat liver plasma membranes (5) and with sera of healthy volunteers. However, with sera of
cancer patients, both the GTP stimulations and GDP inhibitions appeared to be more marked than with sera from the healthy volunteers. The findings provide additional evidence for the uniqueness of the NADH oxidase form present in the sera of cancer patients. When sera were examined for responsiveness to capsaicin, it was found that sera from 10 healthy volunteers selected at random did not respond to capsaicin.
TABLE IV
NADH Oxidase Activities of Sera of Patients with Disorders Other Than Cancer and Response to 1 or 100 mM Capsaicin in DMSO Capsaicin addition
Disorder
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
1 Thrombocytosis 2 Anemia 3 Anemia 4 Myelodysplasic syndrome 5 Cardiac 6 Cardiac 7 Cardiac 8 Cardiac 9 Leukopenia Mean
0.18 0.38 0.80 0.56 0.84 0.68 0.64 0.76 0.68 0.61
0.16 0.38 0.80 0.60 0.80 0.66 0.66 0.72 0.68 0.61
00.02 0.00 0.00 0.04 00.04 00.02 0.02 00.04 0.00 0.00
0.89 1.0 1.0 1.07 0.95 0.97 1.03 0.95 1.0 0.98 { 0.05*
0.18 0.38 0.72 0.60 0.84 0.66 0.66 0.72 0.68 0.61
0.00 0.00 00.08 0.04 0.00 00.02 0.02 00.04 0.00 00.01 { 0.04*
1.0 1.0 0.9 1.07 1.0 0.97 1.03 0.95 1.0 0.99
0.95 1.0 0.95 1.07 0.97 0.97 1.03 0.95 1.0 0.99
Note. All data in nmol/min/100 ml sera. * Not significant.
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Nor did that of 9 patients with disorders other than cancer. In contrast, the NADH oxidase activity of sera from all 45 cancer patients with active diseases did respond to capsaicin. In the majority of the patients the response was inhibition (97% of the assays). In one breast cancer patient, 1 mM capsaicin stimulated the activity. This stimulation was determined not to be a measurement error as a repeat determination with sera from the same patient yielded similar results. Even with this patient, 100 mM capsaicin eventually inhibited the activity. Also stimulated by capsaicin were NADH oxidase activities from sera of a myeloma patient and a leukemia patient, both at 100 mM capsaicin. It is possible that the stimulations also may have diagnostic value as they tended to lie out of the normal range of ú0.9 to õ1.1. Surprisingly, the capsaicin-inhibited NADH oxidase activity was consistently observed over a range of both solid tumors (breast, prostate, colon, lung, ovarian, myeloma, esophageal, and melanoma) as well as with leukemias and lymphomas. Due to variation of absolute specific activities among serum samples, means of NADH oxidase activities of sera from cancer patients (0.7 nmol/min/100 ml sera) were not different statistically from those of apparently healthy laboratory volunteers (0.46 nmol/min/100 ml sera) or patients with disorders other than cancer (0.61 nmol/min/100 ml sera). However, the response to capsaicin was significant when expressed either as an absolute change in rate (D) or as the ratio of the rate in the presence of capsaicin to the rate in the absence of capsaicin. There was no effect of capsaicin with sera from apparently healthy laboratory volunteers. The response to addition of either 1 or 100 mM capsaicin was 0 { 0.02 nmol/min/100 ml sera. With {2 SD as the confidence interval, an absolute D for capsaicin of ú{0.04 was given by at least one of the two capsaicin concentrations for all of the sera samples from cancer patients analyzed. To provide an estimate of sensitivity and specificity of the inhibitory response of the NADH activity to capsaicin, results also were expressed as the ratio of the activities in the presence of capsaicin to the activity in the absence of capsaicin. The values for apparently healthy volunteers were all within the range 0.9 to 1.1. All serum samples from cancer patients exhibited activity ratios (ratio of activity with capsaicin to activity with no capsaicin) of õ0.9 or ú1.1 with at least one of the two capsaicin concentrations. With three sera samples (breast 5, 1 mM capsaicin; myeloma, 100 mM capsaicin; and leukemia 2, 100 mM capsaicin), the cap-
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saicin stimulated. With the breast and myeloma specimens, the stimulations were 50% and well out of the range of variation observed with normal volunteers or disorders other than cancer. With disorders other than cancer, the activity ratios of NADH oxidase in the presence of capsaicin to that in the absence of capsaicin were within the normal range of 0.9 to 1.1 (ú0.9). Overall, the findings suggest that the circulating form of the drug-responsive NADH oxidase from autochthonous tumors is widespread among sera of individual cancer patients. In contrast to the form from culture media conditioned by growth of HeLa cells which was capsaicin-inhibited, the serum form was either inhibited or stimulated by the capsaicin depending on individual patients, although the activity was most often inhibited. In either situation, the results suggest that the shed form of the drug-inhibited NADH oxidase may represent a biochemical characteristic universally associated with malignancy, including not only solid tumors, but leukemias and lymphomas as well. ACKNOWLEDGMENTS We thank Dr. Sarah Sayger of the Purdue University Student Health Center, Drs. Kenneth Pennington and John Ramsey of the Arnett Clinic (Lafayette, IN), Dr. William Jacobsen of St. Elizabeth Hospital (Lafayette, IN), and Drs. Thomas Troeger, Juan C. Garcia, Rafat H. Ansari, and David A. Taber of the Michiana Hematology– Oncology Polyclinic (South Bend, IN), and especially the Hematology Laboratory staff of the Michiana Hematology–Oncology Polyclinic for assistance in collecting serum samples. We are grateful to Connie Chalko, Community Hospital (Plymouth, IN), for coordinating pickup and delivery of serum samples. Financial support was provided in part by Portola Sciences Inc. (Portola Valley, CA) and Phi Beta Psi. The spectrophotometers utilized were provided by an award from Eli Lilly Research Laboratories (Indianapolis, IN).
REFERENCES 1. Morre´, D. J., Chueh, P.-J., and Morre´, D. M. (1995) Proc. Natl. Acad. Sci. USA 92, 1831–1835. 2. Wilkinson, F., Kim, C., Cho, N., Chueh, P.-J., Leslie, S., MoyaCamarena, S., Wu, L.-Y., Morre´, D. M., and Morre´, D. J. (1996) Arch. Biochem. Biophys. 336, 275–282. 3. Brightman, A. O., Wang, J., Miu, R-K., Sun, I. L., Barr, R., Crane, F. L., and Morre´, D. J. (1992) Biochim. Biophys. Acta 1105, 109–117. 4. Bruno, M., Brightman, A. O., Lawrence, J., Werderitsh, D., Morre´, D. M., and Morre´, D. J. (1992) Biochem. J. 284, 625–628. 5. Morre´, D. J., Davidson, M., Geilen, C., Lawrence, J., Flesher, G., Crowe, R., and Crane, F. L. (1993) Biochem. J. 292, 647–653. 6. Morre´, D. J., and Morre´, D. M. (1995) J. Bioenerg. Biomembr. 27, 137–144. 7. Morre´, D. J., Sun, E., Geilen, C., Wu, L-Y., de Cabo, R., Krasagakis, K., Orfanos, C. E., and Morre´, D. M. (1996) Eur. J. Cancer 32A, 1995–2003.
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Vol. 342, No. 2, June 15, pp. 224–230, 1997 Article No. BB970110
NADH Oxidase Activity from Sera Altered by Capsaicin Is Widely Distributed among Cancer Patients D. James Morre´,*,1 Sara Caldwell,* Adrienne Mayorga,* Lian-Ying Wu,† and Dorothy M. Morre´† Departments of *Medicinal Chemistry and Molecular Pharmacology and †Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907
Received December 10, 1996, and in revised form March 19, 1997
A cancer-specific form of NADH oxidase inhibited or stimulated by 1 or 100 mM capsaicin (8-methyl-Nvanillyl-6-noneamide) is present in sera from cancer patients. The capsaicin-inhibited NADH oxidase activity appears to be absent from sera of individuals free of cancer. The capsaicin-inhibited activity is present both in freshly collected sera and in sera stored frozen for varying periods of time. For the latter, an assay was carried out under renaturing conditions in the presence of NADH and reduced glutathione followed by dilute hydrogen peroxide. Inhibition was half maximal at about 1 mM capsaicin. The capsaicin-inhibited activity was found in sera over a broad spectrum of cancer patients including patients with solid cancers (e.g., breast, prostate, lung, ovarian) as well as with leukemias and lymphomas. q 1997 Academic Press Key Words: cancer; serum; NADH oxidase; capsaicin.
Our laboratory described a capsaicin-inhibited NADH oxidase activity associated with plasma membranes of cancer cells grown in culture (1). The EC50 for inhibition of plasma membrane NADH oxidase activity and growth in BT-20 mammary carcinoma cells was about 1 mM (1). A similar capsaicin-inhibited activity was subsequently shown to appear in conditioned culture media of HeLa cells (2) as a shed form of reduced molecular weight. This observation prompted a study to determine if a similar activity might be shed into sera by autochthonous tumors in patients. MATERIALS AND METHODS Sera. Patient sera were from the patient population of the Michiana Hematology–Oncology Clinic (South Bend, IN) and from the 1
To whom correspondence should be addressed at Department of Medicinal Chemistry and Molecular Pharmacology, HANS Life Sciences Research Building, Purdue University, West Lafayette, IN 47907. Fax: (765) 494-4007.
patient population of Arnett Clinic (Lafayette, IN). Sera from normal volunteers were collected by the Michiana Hematology–Oncology Clinic and the Purdue University Health Center. Informed consent was obtained and confidentiality of medical records was assured by assigning a number to each serum sample. Patients were identified only as with active disease (e.g., stage III or stage IV) at the time of collection and as to organ site. Capsaicin (8-methyl-N-vanillyl-6-noneamide) was from Sigma (St. Louis, MO) and dissolved in DMSO.2 The final concentration of DMSO in the assay was 0.1% for 1 mM capsaicin and 0.2% for 100 mM capsaicin. Blanks without capsaicin contained DMSO. Spectrophotometric NADH oxidase assay. NADH oxidase activity was determined at 377C as the disappearance of NADH measured at 340 nm. Activity was measured using a Hitachi U3210 spectrophotometer with stirring and continuous recording over 5-min intervals. The reaction mixture contained 25 mM Tris–Mes buffer (pH 7.2), 1 mM KCN, 150 mM NADH, and 100 ml serum. A millimolar extinction coefficient of 6.22 was used to calculate the rate of NADH disappearance. Frozen samples were assayed by the addition to the complete reaction mixture of 2.5 ml 100 mM reduced glutathione (100 mM final concentration). After 5 to 15 min incubation at 377C, the assay was started by the addition of 2.5 ml 30% hydrogen peroxide (0.03% final concentration) and 2.5 ml 1 mM ATP (final concentration 1 mM). The activity was assayed over 10 to 15 min until a stable steady state was achieved after which 2.5 ml 1 mM capsaicin in DMSO was added (1 mM final concentration). The assay was continued for 10 min, after which 2.5 ml of 100 mM capsaicin in DMSO was added (final concentration of 100 mM) and the assay continued for an additional 10 min. An equivalent amount of DMSO alone was without effect.
RESULTS
Human sera from normal individuals contained NADH oxidase activity (Fig. 1). The activity was sensitive to heating (10 min at 807C). It was proportional to serum concentration and time of incubation (Fig. 1A). The response to NADH was biphasic. The Km for NADH was determined to be 25 mM over the range of 0 to 50 mM NADH and ca. 125 mM over the range 90 to 350 mM NADH (Fig. 1B). 2 Abbreviations used: DMSO, dimethyl sulfoxide; Mes, 4-morpholineethanesulfonic acid; PCMB, p-chloromercuribenzoic acid.
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FIG. 1. Enzymatic characteristics of NADH oxidation by human sera. (A) Proportionality to serum amount and time (inset). (B) Response to NADH concentration.
The activity from sera of cancer patients was stimulated by GTP (Fig. 2) and inhibited by GDP (Fig. 3). The dose–response for pooled sera from cancer patients and from normal volunteers was very similar. The activity was inhibited, as well, by the thiol reagents PCMB (Fig. 4) and N-ethylmaleimide (not shown). The oxidation of NADH was more sensitive to PCMB for cancer sera than for normal sera. The EC50 for sera from cancer patients was 10 nM PCMB, whereas that for sera of healthy volunteers was about ca. 0.1 mM PCMB. A differential response to capsaicin was observed with sera pooled from cancer patients (Fig. 5). NADH oxidase activity was inhibited half maximally by 100 mM capsaicin and maximally by about 1 mM capsaicin. NADH activity of sera pooled from healthy volunteers was unaffected by capsaicin over the same range of capsaicin concentrations that inhibited that of the sera of cancer patients. To begin to investigate the generality of the capsaicin inhibition of NADH oxidase activity of sera from cancer patients, individually collected sera were examined. Values are presented in the order that the data were collected. No values were excluded except for two samples where no rates were obtained. The majority of the samples had been collected within 6 months of assay and were stored frozen. With apparently healthy volunteers, the rate of NADH oxidation varied over a range of 10-fold from
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FIG. 2. Stimulation of NADH oxidase activity of human sera by GTP. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
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FIG. 3. Inhibition of NADH oxidase activity of human sera by GDP. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
FIG. 5. Response to capsaicin of the NADH oxidase activity of pooled sera from cancer patients (open symbols) and from healthy volunteers (solid symbols). Results are averages from three different lots of sera { SD.
FIG. 4. Inhibition of NADH oxidase activity of human sera by PCMB. The solid symbols are for pooled sera from healthy volunteers. The open symbols are for pooled sera from patients with active cancer. Results are averages from three different lots of sera { SD.
0.09 to 0.9 nmol/min/100 ml serum (Table I). The source of this variation is unknown but may relate to the time of holding at room temperature prior to freezing. Activity declined progressively over several hours at room temperature. However, with none of the sera from healthy volunteers did capsaicin affect the activity by more than 5%. Overall, with sera from healthy volunteers, both 1 mM capsaicin and 100 mM capsaicin were without effect. With solid tumors, values were less variable and with three exceptions (breast cancer sample 5 that was stimulated by addition of 1 mM capsaicin and a myeloma sample and a leukemia sample (sample 2) that were stimulated by addition of 100 mM capsaicin), all were inhibited in activity by both 1 and 100 mM capsaicin in DMSO (Table II). Activity was, on average, greater with sera from cancer patients (0.7 nmol/min/100 ml serum) than for healthy volunteers (0.46 nmol/min/100 ml serum). With sera of cancer patients, inhibition was about 22% on average with 1 mM capsaicin in DMSO and about 38% on average with 100 mM capsaicin in DMSO. Inhibition at 100 mM capsaicin was greatest with colon cancer (53%) and least with prostate cancer (26%). The mean inhibited rate of NADH oxidation in the presence of 100 mM capsaicin of 0.4 nmol/min/100 ml serum for sera of cancer patients compared closely to that of sera of apparently healthy laboratory volunteers in the absence of capsaicin (0.46 nmol/min/100 ml serum).
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NADH OXIDASE OF CANCER SERA TABLE I
NADH Oxidase Activities of Sera from Apparently Healthy Laboratory Volunteers and Response to 1 and 100 mM Capsaicin in DMSO Capsaicin addition
Sample
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
1 2 3 4 5 6 7 8 9 10 Mean
0.09 0.11 0.56 0.56 0.12 0.6 0.14 0.92 0.92 0.6 0.46
0.09 0.11 0.56 0.58 0.12 0.58 0.14 0.92 0.96 0.64 0.47
0.00 0.00 0.00 0.02 0.00 00.02 0.00 0.00 0.04 0.04 0.01 { 0.02
1.0 1.0 1.0 1.04 1.0 0.97 1.0 1.0 1.04 1.07 1.01
0.09 0.11 0.58 0.56 0.13 0.58 0.14 0.88 0.92 0.64 0.46
0.00 0.00 0.02 0.00 0.01 00.02 0.00 00.04 0.00 0.04 0.00 { 0.02
1.0 1.0 1.04 1.0 1.08 0.97 1.0 0.96 1.0 1.07 1.01
1.0 1.0 1.02 1.02 1.04 0.97 1.0 0.98 1.02 1.07 1.01
Note. All data in nmol/min/100 ml sera.
With leukemias and lymphomas (Table III), results were similar to those for solid tumors. Serum NADH oxidase averaged 0.7 nmol/min/100 ml serum and was 30% inhibited by 1 mM capsaicin in DMSO and 45% inhibited by 100 mM capsaicin in DMSO. The solid cancers all were represented by metastatic disease, e.g., stage III or IV, and the organ site designations refer to the primary cancer. There was no selection to indicate any general proximate causes such as immunosuppression. We have checked interference by differences in age, sex, and particular medications and none were indicated. Sera collected in parallel to the sera from cancer patients were available for a limited number of disorders other than cancer. Results are summarized in Table IV. Although mean values for NADH oxidase activity were, on average, higher than for normal, no response to capsaicin outside of the normal range was observed with any of the sera. DISCUSSION
Our laboratory previously reported a hormone- and growth factor-stimulated NADH oxidase activity of the mammalian plasma membrane (3). The activity was present as well in plasma membranes of rat hepatomas but was no longer growth factor and hormone responsive (4). The activity in plasma membranes from rat liver was stimulated by GTP and other nucleoside triphosphates and inhibited by GDP (5). The activity from hepatoma and HeLa cell membranes, but not that from rat liver, was inhibited by thiol reagents including PCMB and N-ethylmaleimide (6). A further characteristic of the NADH oxidase activity of plasma mem-
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branes from HeLa and other transformed cell lines was its ability to be inhibited by capsaicin (8-methyl-N-vanillyl-6-noneamide) (1). The ability of capsaicin to inhibit NADH oxidase activity correlated with the ability of NADH oxidase to induce apoptosis and prevent the growth of tumor cells (1). Capsaicin inhibited NADH oxidase activity and growth of human cervical carcinoma (HeLa), adenocarcinoma, ovarian carcinoma, HL-60, K-562, and melanoma cells but not of mammary epithelia or melanocytes (1, 7). Also not inhibited was NADH oxidation by plasma membrane vesicles from rat liver, normal rat kidney cells, and Chinese hamster ovary cells. A capsaicin-inhibited NADH oxidase activity similar to that observed with plasma membranes isolated from HeLa cells was found to be present in culture media conditioned by the growth of HeLa cells (2). This activity was identified, based on antisera raised against the protein from culture media, to represent a shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane. The shed form of the activity showed the same response to capsaicin and to thiol reagents as the plasma membraneassociated form. These findings led us to seek evidence for a shed form of the capsaicin-inhibited NADH oxidase in sera of patients with active cancer. Human sera did contain NADH oxidase activity. The Km for NADH and response to guanine nucleotides was similar to that of the plasma membrane-associated form (3, 5). The NADH oxidase activity of serum from cancer patients with active disease also exhibited a greater sensitivity to the thiol reagent PCMB than was observed with sera of
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TABLE II
NADH Oxidase Activities of Sera from Cancer Patients with Active Disease and Response to 1 and 100 mM Capsaicin in DMSO Capsaicin addition
Cancer Breast 1 2 3 4 5 6 7 8 9 10 Mean Prostate 1 2 3 4 5 6 7 8 9 10 Mean Colon 1 2 3 4 5 Mean Lung 1 2 3 4 5 Mean Other Ovarian Myeloma Esophageal Ovarian Melanoma Mean Note. All data in * Significant P ** Significant P *** Significant P † Significant P †† Significant P ††† Significant P ‡ Significant P ‡‡ Significant P
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None (1)
1 mM (2)
Ratio 2/1
100 mM (3)
0.38 0.84 0.72 1.12 0.32 0.72 1.00 0.68 0.60 0.32 0.67
0.26 0.68 0.50 0.96 0.48 0.64 0.28 0.56 0.46 0.16 0.50
1.20 1.00 0.68 1.04 1.28 1.16 0.54 0.40 1.08 1.28 0.97
Ratio 3/1
Average ratio
00.12 00.16 00.22 00.16 0.16 00.08 00.72 00.12 00.14 00.16 00.17 { 0.18*
0.68 0.81 0.69 0.86 1.5 0.89 0.28 0.82 0.77 0.50 0.78
0.28 0.52 0.48 0.16 0.20 0.44 0.60 0.56 0.36 0.14 0.37
00.10 00.32 00.24 00.96 00.12 00.28 00.40 00.12 00.24 00.18 00.30 { 0.25**
0.74 0.62 0.67 0.14 0.63 0.61 0.60 0.82 0.60 0.44 0.59
0.71 0.71 0.68 0.5 (1.07) 0.75 0.44 0.82 0.69 0.47 0.69
0.88 0.80 0.56 0.88 1.16 0.96 0.44 0.32 0.96 1.04 0.80
00.32 00.20 00.12 00.16 00.12 00.20 00.10 00.08 00.12 00.24 00.17 { 0.7†
0.73 0.8 0.82 0.85 0.91 0.83 0.81 0.8 0.89 0.81 0.83
0.68 0.76 0.52 0.88 0.92 0.68 0.48 0.36 0.68 0.96 0.69
00.52 00.24 00.16 00.16 00.36 00.48 00.06 00.04 00.40 00.96 00.28 { 0.17†
0.57 0.76 0.76 0.85 0.72 0.59 0.89 0.9 0.63 0.75 0.74
0.65 0.78 0.79 0.85 0.81 0.71 0.85 0.85 0.76 0.78 0.78
0.46 0.22 0.20 1.12 0.48 0.50
0.22 0.06 0.04 0.92 0.38 0.32
00.24 00.18 00.16 00.20 00.10 00.18 { 0.05††
0.48 0.27 0.2 0.82 0.79 0.51
0.16 0.00 0.18 0.32 0.38 0.21
00.30 00.22 00.02 00.80 00.10 00.29 { 0.31†††
0.35 0 0.9 0.29 0.79 0.47
0.41 0.13 0.55 0.55 0.79 0.49
0.72 0.68 0.72 0.68 0.56 0.67
0.64 0.48 0.68 0.52 0.50 0.56
00.08 00.20 00.04 00.16 00.06 00.11 { 0.07***
0.89 0.71 0.94 0.76 0.89 0.84
0.56 0.18 0.56 0.56 0.44 0.46
00.16 00.50 00.16 00.12 00.12 00.21 { 0.16††
0.78 0.26 0.78 0.82 0.79 0.69
0.83 0.49 0.86 0.79 0.84 0.76
1.08 0.16 1.20 0.46 0.56 0.69
0.92 0.12 0.72 0.32 0.48 0.51
00.16 00.04 00.48 00.14 00.08 00.18 { 0.17‡
0.85 0.75 0.6 0.69 0.86 0.75
0.96 0.24 0.28 0.28 0.40 0.43
00.12 0.08 00.92 00.18 00.16 00.26 { 0.35‡‡
0.89 1.5 0.23 0.61 0.71 0.79
0.87 (1.13) 0.41 0.65 0.79 0.77
D
nmol/min/100 ml sera. õ 0.0056. õ 0.00014. õ 0.0008. õ 0.0001. õ 0.001. õ 0.0074. õ 0.0031. õ 0.03.
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D
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NADH OXIDASE OF CANCER SERA TABLE III
NADH Oxidase Activity of Sera from Patients with Active Leukemia or Lymphomas and Response to 1 or 100 mM Capsaicin in DMSO Capsaicin addition
Disease Leukemia 1 Granulocytic 2 Granulocytic 3 Granulocytic 4 Lymphocytic 5 Lymphocytic Mean Lymphoma (non-Hodgkin’s) 1 2 3 4 5 Mean
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
0.18 0.30 1.12 0.88 0.64 0.63
0.12 0.20 0.84 0.72 0.40 0.46
00.06 00.10 00.28 00.16 00.24 00.17 { 0.09*
0.67 0.67 0.75 0.82 0.63 0.71
0.00 0.32 0.72 0.68 0.36 0.42
00.18 0.02 00.40 00.20 00.28 00.21 { 0.14**
0 1.07 0.64 0.77 0.56 0.61
0.33 (0.87) 0.69 0.79 0.59 0.65
0.48 1.04 0.68 1.08 0.66 0.79
0.26 0.72 0.52 0.68 0.56 0.55
00.22 00.32 00.16 00.40 00.10 00.24 { 0.12*
0.54 0.69 0.76 0.63 0.85 0.69
0.06 0.52 0.52 0.44 0.44 0.40
00.42 00.52 00.16 00.64 00.22 00.39 { 0.20*
0.13 0.5 0.76 0.41 0.67 0.49
0.33 0.59 0.76 0.52 0.76 0.59
Note. All data in nmol/min/100 ml sera. * Significant P õ 0.0001. ** Significant P õ 0.0003.
apparently healthy volunteers. A similar phenomenon was observed with the NADH oxidase activity of HeLa plasma membranes compared to that of rat liver. The shed form of the activity found in sera of cancer patients also was stimulated by GTP and inhibited by GDP. A similar response was seen previously with the NADH oxidase of rat liver plasma membranes (5) and with sera of healthy volunteers. However, with sera of
cancer patients, both the GTP stimulations and GDP inhibitions appeared to be more marked than with sera from the healthy volunteers. The findings provide additional evidence for the uniqueness of the NADH oxidase form present in the sera of cancer patients. When sera were examined for responsiveness to capsaicin, it was found that sera from 10 healthy volunteers selected at random did not respond to capsaicin.
TABLE IV
NADH Oxidase Activities of Sera of Patients with Disorders Other Than Cancer and Response to 1 or 100 mM Capsaicin in DMSO Capsaicin addition
Disorder
None (1)
1 mM (2)
D
Ratio 2/1
100 mM (3)
D
Ratio 3/1
Average ratio
1 Thrombocytosis 2 Anemia 3 Anemia 4 Myelodysplasic syndrome 5 Cardiac 6 Cardiac 7 Cardiac 8 Cardiac 9 Leukopenia Mean
0.18 0.38 0.80 0.56 0.84 0.68 0.64 0.76 0.68 0.61
0.16 0.38 0.80 0.60 0.80 0.66 0.66 0.72 0.68 0.61
00.02 0.00 0.00 0.04 00.04 00.02 0.02 00.04 0.00 0.00
0.89 1.0 1.0 1.07 0.95 0.97 1.03 0.95 1.0 0.98 { 0.05*
0.18 0.38 0.72 0.60 0.84 0.66 0.66 0.72 0.68 0.61
0.00 0.00 00.08 0.04 0.00 00.02 0.02 00.04 0.00 00.01 { 0.04*
1.0 1.0 0.9 1.07 1.0 0.97 1.03 0.95 1.0 0.99
0.95 1.0 0.95 1.07 0.97 0.97 1.03 0.95 1.0 0.99
Note. All data in nmol/min/100 ml sera. * Not significant.
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Nor did that of 9 patients with disorders other than cancer. In contrast, the NADH oxidase activity of sera from all 45 cancer patients with active diseases did respond to capsaicin. In the majority of the patients the response was inhibition (97% of the assays). In one breast cancer patient, 1 mM capsaicin stimulated the activity. This stimulation was determined not to be a measurement error as a repeat determination with sera from the same patient yielded similar results. Even with this patient, 100 mM capsaicin eventually inhibited the activity. Also stimulated by capsaicin were NADH oxidase activities from sera of a myeloma patient and a leukemia patient, both at 100 mM capsaicin. It is possible that the stimulations also may have diagnostic value as they tended to lie out of the normal range of ú0.9 to õ1.1. Surprisingly, the capsaicin-inhibited NADH oxidase activity was consistently observed over a range of both solid tumors (breast, prostate, colon, lung, ovarian, myeloma, esophageal, and melanoma) as well as with leukemias and lymphomas. Due to variation of absolute specific activities among serum samples, means of NADH oxidase activities of sera from cancer patients (0.7 nmol/min/100 ml sera) were not different statistically from those of apparently healthy laboratory volunteers (0.46 nmol/min/100 ml sera) or patients with disorders other than cancer (0.61 nmol/min/100 ml sera). However, the response to capsaicin was significant when expressed either as an absolute change in rate (D) or as the ratio of the rate in the presence of capsaicin to the rate in the absence of capsaicin. There was no effect of capsaicin with sera from apparently healthy laboratory volunteers. The response to addition of either 1 or 100 mM capsaicin was 0 { 0.02 nmol/min/100 ml sera. With {2 SD as the confidence interval, an absolute D for capsaicin of ú{0.04 was given by at least one of the two capsaicin concentrations for all of the sera samples from cancer patients analyzed. To provide an estimate of sensitivity and specificity of the inhibitory response of the NADH activity to capsaicin, results also were expressed as the ratio of the activities in the presence of capsaicin to the activity in the absence of capsaicin. The values for apparently healthy volunteers were all within the range 0.9 to 1.1. All serum samples from cancer patients exhibited activity ratios (ratio of activity with capsaicin to activity with no capsaicin) of õ0.9 or ú1.1 with at least one of the two capsaicin concentrations. With three sera samples (breast 5, 1 mM capsaicin; myeloma, 100 mM capsaicin; and leukemia 2, 100 mM capsaicin), the cap-
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saicin stimulated. With the breast and myeloma specimens, the stimulations were 50% and well out of the range of variation observed with normal volunteers or disorders other than cancer. With disorders other than cancer, the activity ratios of NADH oxidase in the presence of capsaicin to that in the absence of capsaicin were within the normal range of 0.9 to 1.1 (ú0.9). Overall, the findings suggest that the circulating form of the drug-responsive NADH oxidase from autochthonous tumors is widespread among sera of individual cancer patients. In contrast to the form from culture media conditioned by growth of HeLa cells which was capsaicin-inhibited, the serum form was either inhibited or stimulated by the capsaicin depending on individual patients, although the activity was most often inhibited. In either situation, the results suggest that the shed form of the drug-inhibited NADH oxidase may represent a biochemical characteristic universally associated with malignancy, including not only solid tumors, but leukemias and lymphomas as well. ACKNOWLEDGMENTS We thank Dr. Sarah Sayger of the Purdue University Student Health Center, Drs. Kenneth Pennington and John Ramsey of the Arnett Clinic (Lafayette, IN), Dr. William Jacobsen of St. Elizabeth Hospital (Lafayette, IN), and Drs. Thomas Troeger, Juan C. Garcia, Rafat H. Ansari, and David A. Taber of the Michiana Hematology– Oncology Polyclinic (South Bend, IN), and especially the Hematology Laboratory staff of the Michiana Hematology–Oncology Polyclinic for assistance in collecting serum samples. We are grateful to Connie Chalko, Community Hospital (Plymouth, IN), for coordinating pickup and delivery of serum samples. Financial support was provided in part by Portola Sciences Inc. (Portola Valley, CA) and Phi Beta Psi. The spectrophotometers utilized were provided by an award from Eli Lilly Research Laboratories (Indianapolis, IN).
REFERENCES 1. Morre´, D. J., Chueh, P.-J., and Morre´, D. M. (1995) Proc. Natl. Acad. Sci. USA 92, 1831–1835. 2. Wilkinson, F., Kim, C., Cho, N., Chueh, P.-J., Leslie, S., MoyaCamarena, S., Wu, L.-Y., Morre´, D. M., and Morre´, D. J. (1996) Arch. Biochem. Biophys. 336, 275–282. 3. Brightman, A. O., Wang, J., Miu, R-K., Sun, I. L., Barr, R., Crane, F. L., and Morre´, D. J. (1992) Biochim. Biophys. Acta 1105, 109–117. 4. Bruno, M., Brightman, A. O., Lawrence, J., Werderitsh, D., Morre´, D. M., and Morre´, D. J. (1992) Biochem. J. 284, 625–628. 5. Morre´, D. J., Davidson, M., Geilen, C., Lawrence, J., Flesher, G., Crowe, R., and Crane, F. L. (1993) Biochem. J. 292, 647–653. 6. Morre´, D. J., and Morre´, D. M. (1995) J. Bioenerg. Biomembr. 27, 137–144. 7. Morre´, D. J., Sun, E., Geilen, C., Wu, L-Y., de Cabo, R., Krasagakis, K., Orfanos, C. E., and Morre´, D. M. (1996) Eur. J. Cancer 32A, 1995–2003.
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