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“Big” insulin and the treatment of an islet cell carcinoma with streptozotocin
“Big” Insulin and the Treatment of an Islet Cell Carcinoma With Streptozotocin By Margaret
J. Pearson,
R. G. Larkins,
Total plasma immunoreactive insulin (1RI) and percentage “big” insulin (proinsulinlike material) were measured in a patient with an inoperable islet cell carcinoma who did not improve clinically after streptozotocin administration. Prior to the streptozotocin, the total IRI rose with administration of tolbutamide, and the percentage of “big” insulin remained constant. After intravenous glucose there was little change in plasma IRI, but a substantial rise in the percentage of “big” insulin occurred. The admin-
and F. I. R. Martin
istration of streptozotocin was acutely with hypoglycemia, a associated slight increase in total IRI, and no change in the percentage “big” insulin. With chronic administration and extension of the disease, the total IRI and percentage “big” insulin rose progressively. It is concluded that the percentage “big” insulin rises as loss of tumor differentiation occurs and that changes in the percentage of “big” insulin after streptozotocin may give an early indication of likely response to streptozotocin.
S
TREPTOZOTOCIN is an antibiotic isolated from Streptomyces achromogenes1~2 that has cytotoxic properties specific for the P-cells of the pancreas.3-5 Its use in the treatment of several patients with metastatic islet cell carcinoma has been reported, with excellent symptomatic relief and regression of the tumor in some instances 6-11 but only partial benefit in others.12s13 Blackard et aL7 investigated qualitative aspects of plasma insulin in a patient following successful treatment with streptozotocin. They found that the high proportion of “big” insulin (proinsulinlike material) seen initially after oral glucose was reduced after therapy, despite minimal change in plasma immunoreactive insulin (IRI) concentrations. In contrast, the present report describes changes in “big“ insulin and IRI in a patient who did not respond to streptozotocin. It is hoped that some predictive factors regarding potential response to streptozotocin may emerge from reports such as this. CASE REPORT A 56-yr-old man presented to another hospital with a 2-mo history of abdominal pain, anorexia, weight loss, and diarrhea. The symptoms bore no relationship to meals or fasting. The only symptom directly attributable to hypoglycemia was a recent preference for sweet foods for breakfast. The liver was hard and irregular and extended 8 cm below the costal margin.
From the Department of Biochemistry and Endocrinology, The Royal Melbourne Hospital, Melbourne, Auslralia. Received for publication October 15, 1971. Margaret J. Pearson, M.Sc.: Endocrine Biochemist, Department of Biochemistry, Royal Melbourne Hospital, Melbourne, AustraZia. R. G. Larkins, M.B.B.S., M.R.A.C.P.: A&slant Endocrinologist, Royal Melbourne Hospital, and Associate, University of Melbourne Department of Medicine, Royal Melbourne Hospital, Melbourne, Australia. F. I. R. Martin, M.D., F.R.A.C.P. : Endocrinologist, Royal Melbourne Hospital, and Senior Associate, University of Melbourne Department of Medicine, Royal Melbourne Hospital, Melbourne, Australia. Metabolism,
Vol. 21, No. 6 (June), 1972
661
552 Laparotomy
PEARSON,
revealed
a large
tumor
mass
LARKINS,
in the body of the pancreas,
AND MARTIN
with
multiple
hepatic metastases and a distended gall bladder. A prophylactic cholecvstoieiimostomv was performed. Six hours postoperatively, after awaking from the anesthetic, the patient lansed into a deep coma. Blood sugar at that time was 12 mg/lOO ml. and the patient responded promptly to intravenous dextrose. Histologic examination of the operative biopsy revealed cords and clumps of cells, varied in size and shape, often with large and dark nuclei and numerous mitoses, consistent with an islet cell carcinoma of the pancreas. Plasma IRI levels ranging between 78 uU/ml and 142 pUlm1 in the presence of hypoglycemia confirmed this diagnosis. Therapy with corticosteroids (dexamethasone 16 mg daily) and diazoxide (800 mg daily) was ineffective in controlling hypoglvcemia, and a continuous intravenous infusion of 20% fructose was required. This resulted in the development of lactic acidosis fplasma lactate 46 mg/lOO ml, normal 5-15 mgboo ml and serum bicarbonate 7 meq/liter, normal 20-30 meq/ liter), so that 10% dextrose was substituted. Three weeks postoperatively a combination of glucagon (8 mg infused intravenously daily), dexamethasone (12 mg infused intravenously daily), and diazoxide (100 mg orally at 6-hr intervals) provided partial control. However, when intravenous fluids and glucagon injections were discontinued occasional severe hypoglycemic episodes recurred, despite the maximum tolerated doses of diazoxide and dexamethasone. Peripheral edema developed, and bendroflumethiazide, .S mg daily, was added. In view of the failure to adequately rontrol the hypoglycemia, streptozotocin (Upjohn International Inc.) was administered s wk postoperatively. Two doses of 1.5 g, made up in citrate buffer pH 4.4 immediately before injection, were given intravenously, with 7 days between doses. Each dose was followed by nausea and vomiting, but not by hypoglycemia. For 2 wk there was symptomatic improvement, but then hypoglycemia recurred. A further 3 g of streptozotocin i.v. resulted in severe hypoglycemic episodes immediately and z hr and 12 hr later, in spite of continuous infusion of fructose. After 2 wk more in which the patient was comparatively well the symptoms recurred, and the liver had obviously increased in size. A further 1.5 g of streptozotocin was given intravenously, and 2 days later 1.5 g was infused into the celiac artery. Intravenous fructose was again required to control acute hypoglycemia. Pulmonary and periphera1 edema developed that was unresponsive to diuretic therapy, digoxin, and aminophylline, and the patient died from pulmonary edema 28 hr after the last dose of streptozotocin. Liver function tests throughout the patient’s illness were abnormal with markedly elevated glutamic-oxaloacetic transaminase and alkaline phosphatase; no further change occurred after streptozotocin. Liver scan 1 mo postoperatively, prior to the streptozotocin, showed evidence of hepatic metastases, and 2 mo later, just prior to death, a repeat scan showed further growth of the metastatic lesions. Blood urea was normal and did not rise following streptozotocin. There was no evidence of renal tubular dysfunction. Serum potassium fell to 3.5 meq/liter (normal 3.5-5.5 meq/liter) whiIe the patient was on diuretic therapy. The hemoglobin was slightly reduced initially but did not fall following streptozotocin. Serum calcium and immunoassayable gastrin were normal, and s-hydroxyindoleacetic acid was not detected in the urine. Autopsy revealed a 3 cm diameter white necrotic tumor in the head of the pancreas. Metastatic tumor was present in portal lymph nodes and in the massively enlarged liver. Gross postmortem autolysis made estimation of the degree of necrosis in the tumor impossible.
MATERIALS
AND METHODS
Blood sugar, plasma IRI, and the percentage of “big” insulin (measured as immunoreactive insulin) were determined during both an intravenous glucose tolerance test 7.50 mg/kg) and in intravenous tolbutamide tolerance test (1 g) performed on successive days 1 wk prior to streptozotocin administration. Neither test was performed under basal conditions, as hypoglycemic episodes prevented this. An infusion of 5% dextrose at a constant rate of 1 liter/l2 hr was continued up to 1 hr before the time of the glucose injection in
TREATMENT OF CELL CARCINOMA WITH STREPTOZOTOCIN
5(
-
INSULIN.
.__
PRO-INSULIN
3c
Fig. 1. Solid line represents standard curve obtained by displacement of 126l-labeled insulin from antibody by unlabeled insulin. Solid dots show points obtained by serial dilution of freeze-dried extracts of “little” insulin fraction obtained (see text). Interrupted line represents standard curve obtained for proinsulin, and partially open dots show points obtained by serial dilution of freeze-dried extracts of “big” insulin fraction obtained.
20
10
o.; COLD
STANDARD
ADDED
hglml)
Blood sugars were measured by the Autoanalyser (Technicon) ferricyanide method,14 and plasma IRI by the charcoal method of Herbert et al.15 as modified by Pearson et al,16 using 125-I insulin and a pork insulin standard (Lilly, Lot P.J. 5589). Insulin sensitivity (KIw) was determined as previously described.17 Plasma “big” and “little” insulin were assessed by a modification of the method of Roth et al.,18 as described by Blackard et al.,’ except that the buffer used was 0.01 M Verona1 the glucose tolerance test, and the same infusion was continued at a constant rate throughout the tolbutamide tolerance test. The plasma IRI and percentage “big” insulin were monitored acutely and chronically following the administration of streptozotocin. The values were never obtained under truly basal conditions due to the necessity of frequent (2-hr intervals) glucose feeding during the night prior to the tests.
PRO-INSULIN 0
FRACTION NUMBER
INSULIN
Fig. 2. Elution pattern of porcine proinsulin and porcine insulin standard preparations on Sephadex G-50 column.
554
I.R.I.
-uhl
PEARSON,
LARKINS.
AND MARTIN
as. Xet h&
II
I
TIME AFTER-Ii.
GLUCOSi
(MN)
A
TIME
AFTER
t.v.
TOLBUTAMlOE
Fig. 3. (A) Blood sugar (B.S.) shown by continuous line, total I.R.I. shown by gray columns, and percentage “big” insulin (B.I.) shown by black columns, after intravenous administration of 750 mg/kg glucose i.v. (B) Blood sugar, total I.R.I., and percentage “big” insulin after 1 g tolbutamide i.v. Symbols are same as in Fig. 3A.
(mid
B
buffer pH 7.4 containing 0.2% sodium azide and that 2 ml of plasma was used for each sample. “Big” insulin isolated in this way is probably a crude preparation of proinsulin. In preliminary experiments, “big” insulin and “little” insulin obtaned from plasma by this method were characterized by comparison with standard porcine proinsulin (Lilly, Lot 6151082&46.2) and insulin. The immunologic behavior on serial dilution of freeze-dried fractions (Fig. 1) and the elution pattern on Sephadex G-50 (Fig. ?-) confirmed the differing molecular weights and immunoreactivity of “big” and “little” insulin. In the subsequent text, percentage “big” insulin refers to the insulin eluting as “big” insulin on Sephadex G-50 read from the insulin standard curve expressed as a percentage of the total irnmunoreactive insulin in the whole serum, again read from the insulin standard curve.
RESULTS Intravenous glucose produced only a small increase in plasma insulin, although the percentage of “big” insulin increased from 14% at fasting to 45% 4.5 min after the glucose injection (Fig. 3A). In contrast, intravenous tolbutamide produced a marked increase in plasma IRI accompanied by a prolonged and exaggerated fall in blood sugar, occurring after an initial unexplained rise. The percentage contribution of “big” insulin to the total IRI after tolbutamide did not change significantly (Fig. 3B), reflecting a considerable output of “big” insulin as well as “little” insulin. However, the percentage “big” insulin at the commencement of this test was considerably higher than the initial level during the glucose tolerance test on the previous day, perhaps because of the continuous slow glucose infusion.
TREATMENT
OF CELL CARCINOMA
A
Fig. 4. (A) Blood sugar (B.S.) (continuous line), total I.R.I. (gray columns), and percentage “big” insulin (B.I.) (black columns) in 4 hr after single dose of 1.5 g streptozotocin intravenously, given at time indicated by arrow. (B) Total I.R.I. (gray columns) and percentage “big” insulin (B.I.) (black columns) over 2 mo. during which streptozotocin was given. Arrows represent doses of streptozotocin.
B
WITH STREPTOZOTOCIN
555
TIME
TIME
Initially, insulin sensitivity was less than normal (KITT 1.2, normal > 2.4), and after 6 g of streptozotocin had been given this remained unchanged. Administration of a total of 9 g of streptozotocin over an 8-wk period did not appear to significantly affect either plasma IRI or the percentage “big” insulin. The levels of both increased in parallel with clinical progression of the tumor, the plasma IRI rising to over 500 rU/ml, and the percentage “big” insulin reaching 80% (Fig. 4B). All readings were obtained under similar conditions, with 2-hr glucose feeds throughout the previous night up to 2 hr before the test was taken. After the second test shown on Fig. 4B, the patient was also receiving 400 mg diazoxide daily, 6-8 mg dexamethasone daily, and 5 mg bendroflumethiazide daily. These agents should have decreased the plasma insulin, but a progressive rise occurred in spite of them. Similarly, streptozotocin appeared to have little acute effect on either the plasma IRI or “big” insulin over a 5-hr period immediately following the intravenous injection of 1.5 g streptozotocin (Fig. 4A). Although the blood sugar dropped precipitously to 10 mg/100 ml 2% hr after the injection, there was only a slight increase in the plasma IRI (460-510 pU/ml) and no significant change in the percentage “big” insulin (62-60%) at the time of maximal hypoglycemia.
PEARSON,
556
LARKINS,
AND MARTIN
DISCUSSION
This patient with a metastatic islet cell carcinoma showed high plasma IRI levels with a proportion of “big” insulin that increased from normal to extremely high levels during the course of the disease. These findings are compatible with previous reports, both of norma120s21 and high21-25 levels of proinsulin or proinsulinlike material in patients with islet cell tumors. The tumor showed a varied pattern of response to stimuli. The proportion of “big” insulin increased following glucose, as has been reported for normal subjects,lg but remained virtually unchanged after tolbutamide. However, this represented a large increase in the absolute quantity of “big” insulin when the rise in total IRI was taken into account. Gutman et al. found in a patient with a benign insulinoma that tolbutamide apparently released “big” insulin with 71% of the plasma IRI in the proinsulinlike fraction after tolbutamide and only 5% after oral glucose. 21 In contrast, Gorden and Roth reported a patient with an islet cell carcinoma in whom the percentage of proinsulinlike material fell from 37 to 3% 15 min after intravenous toIbutamide.25 Similarly, the patient of Melani et al. with a benign tumor showed a fall in the proportion of proinsulin following tolbutamide,24 and there was little change in the absolute proinsulin concentration following tolbutamide in the patient of Taylor et a1.,8 who like our patient had a malignant lesion. In contrast to the majority of reported cases, this patient apparently did not respond to streptozotocin. The dose given was similar to that used by the majority of workers, 6,7*11 but much less than the total dose of 30 g given by Stanley et a1.g The characteristic hypoglycemic episodes following the administration of the drug confirmed its activity. Stanley et al9 suggested that injection into the cehac artery was the most effective route, although this increases the danger of nephrotoxicity due to inadvertent administration into the renal artery. The last dose given to this patient was injected into the celiac artery, but survival after this was too short to assess either its efficacy or side effects. The refractory pulmonary edema that caused the patient’s death was probably due to the combination of intravenous fluid, diazoxide, and corticosteroids, although a possible role of streptozotocin could not be discounted. The relative ineffectiveness of streptozotocin in the present case may have been due to the massive size of the tumor or to the particular abnormality of the enzymic constitution of the tumor cells. The mechanism of the P-cytotoxic effect of the drug is not known. TayIor 26 demonstrated a direct inhibitory effect on insulin synthesis, but in practice a reduction in plasma insulin is not always found even in patients who respond symptomatically to the drug.’ It has also been postulated27‘2s that streptozotocin interferes with nicotinamide adenine dinucleotide formation in the P-cell, but it is not known whether an alteration in P-cell pyridine nucleotide content would result in diabetogenecity. After acute administration of streptozotocin in the present case, there was no striking alteration in the plasma insulin concentration or in the percentage of “big” insulin; in spite of this, marked hypoglycemia occurred (Fig. 4A).
TREATMENT
OF CELL CARCINOMA
WITH STREPTOZOTOCIN
557
This may have been due to altered peripheral sensitivity to insulin or to change in biologic but not immunologic reactivity of the “little” insulin released. Alternatively, the slight increase in total IRI may have accounted for the hypoglycemia. Altered sensitivity to insulin after streptozotocin appears unlikely, as there was no significant change in KI.L.~. after chronic administration. To our knowledge, the only other published study reporting changes in proinsulin after streptozotocin is that of Blackard et al7 In their patient, clinical improvement coincided with a fall in percentage of “big” insulin found after oral glucose, although no information on fasting levels was given. It is likely that the increase in the percentage “big” insulin we observed was a manifestation of progression of the disease with further loss of differentiation of the tumor cells. This supports the suggestion7 that the proportion of “big” insulin may be a marker reflecting the abnormality in insulin biosynthesis and release. This is in accord with the observation of Gutman et a1.21 that the highest percentage of proinsulinlike material occurs in malignant islet cell tumors. The rise in percentage “big” insulin occurring despite streptozotocin may be an early indication of insensitivity to this agent. It is hoped that further studies of IRI and proinsulinlike material in plasma, together with their acute response to stimuli such as tolbutamide and glucose, may define the group of patients with inoperable islet cell carcinomas most likely to respond to streptozotocin. ACKNOWLEDGMENT The help of Dr. B. King, who initially referred the patient, and Dr. L. Murton, who assisted in obtaining the clinical data, is gratefully acknowledged. The streptozotocin was supplied by Upjohn International Inc., and the proinsulin by Eli LiIly. Dr. J. Hansky of the Department of Medicine, Monash University, performed the gastrin assay.
REFERENCES 1. Herr, R. R., Jahnke, H. K., and Argoudelis, A. D.: The structure of streptozotocin. J. Amer. Chem. Sot. 89:4808, 1967. 2. Vavra, J. J., DeBoer, C., Dietz, A., Hanka, L. J., and Sokolski, W. T.: Antibiotics Annual. New York, Antibiotica, 1960, 230. 3. Junod, A., Lambert, A. E., Orci, L., Pickett, R., Gonet, A. E., and Renold, A. E.: Studies on the diabetogenic action of streptozotocin. Proc. Sot. Exp. Biol. Med. 126: 201,1967. 4. Pitkin, R. M., and Reynolds, W. A.: Diabetogenic effects of streptozotocin in rhesus monkeys. Diabetes 19 :85,1970. 5. Rakieten, N., Rakieten, M. L., and Nadkarni, M. V.: Studies on the diabetogenie action of streptozotocin. Cancer Chemother. Rep. 29:91, 1963. 6. Murray-Lyon, I. M., Eddleston, A. L. W. F., Williams, R., Brown, M., Hogbin,
B. M., Bennett, A., Edwards, J. C., and Taylor, K. W.: Treatment of multiplehormone producing malignant islet-cell tumour with streptozotocin. Lancet 2 :895, 1968. 7. Blackard, W. G., Garcia, A. R., and Brown, C. L.: Effect of streptozotocin on qualitative aspects of plasma insulin in a patient with a malignant islet cell tumour. J. Clin. Endocr. 31:215, 1970. 8. Taylor, S. G., Schwartz, T. B., Zannini, T. J., and Ryan, W. G.: Streptozotocin therapy for metastatic insulinoma. Arch. Intern. Med. (Chicago) 126:654, 1970. 9. Stanley, N. N., Marks, V., Kreel, L., and McIntyre, N.: Streptozotocin treatment of malignant islet cell tumour. Brit. Med. J. 3:562,1970. 10. Arnould, Y., Ooms, H. A., and Bastenie, I’. A.: Treatment of insulinoma with streptozotocin. Lancet 1:1210, 1969.
558 11. Sadoff, L.: Effects of streptozotocin in a patient with islet cell carcinoma. Diabetes 18:675,1969. 12. Livingston, R. B., and Carter, S. K.: Chemotherapy Fact Sheet, Program Analysis Branch, Chemotherapy, National Cancer Institute, Bethesda, Md. 1969, p 1. 13. Ennis, G.: Personal communication. 14. Hoffman, W. A.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:.51, 1937. 15. Herbert, V., Lau, K., Gottlieb, C. W., and Bleicher, S. J. : Coated charcoal immunoassay of insulin. J. Clin. Endocr. 25:1375, 1965. 16. Pearson, M. J., Fullerton, M. J., Martin, F. I. R., and Melick, R.: An assessment of the charcoal immunoassay of insulin. Proc. Aust. Ass. Clin. Biochem. 1:315, 1968. 17. Martin, F. I. R., Pearson, M. J., and Stocks, A. E.: Glucose tolerance and insulin insensitivity. Lancet 1:1285, 1968. 18. Roth, J. Gorden, P., and Pastan, I.: “Big insulin”: a new component of plasma insulin detected by immunoassay. Proc. Nat. Acad. Sci. USA 61:138, 1968. 19. Melani, F., Rubenstein, A. H., and Steiner, D. F.: Human serum proinsulin. J. Clin. Invest. 49:497, 1970. 20. Gutman, R. A., Lazarus, N. R., Penhos, J. C., Recant, L., and Fajans, S. S.: Proinsulin (P.I.) and proinsulin-like material (PI-LM) in serum of patients with islet cell tumours. Diabetes X3:360, 1969. 21. -, -, -, Fajans, S., and Recant, L.: Circulating proinsulin-like material in func-
PEARSON,
LARKINS,
AND MARTIN
tioning insulinomas. New Eng. J. Med. 284: 1003,197l. 22. Lazarus, N. R., Tanese, T., Gutman, I’. A., and Recant, L.: Synthesis and release of proinsulin and insulin by human insulinoma tissue. J. Clin. Endocr. 30:273, 1970. 23. Goldsmith, S. J., Yalow, R. S., and Berson, S. A.: Significance of human plasma insulin sephadex fractions. Diabetes 18:834, 1969. 24. Melani, F., Ryan, W. G., Rubinstein, A. H., and Steiner, D. F.: Proinsulin secretion by a pancreatic beta-cell adenoma. New Eng. J. Med. 83713, 1970. 25. Gorden, P., and Roth, J.: Plasma insulin: fluctuations in the “big” insulin component in man after glucose and other stimuli. J. Clin. Invest. 48:2225, 1969. 26. Taylor, K. W.: Regulation of insulin synthesis. In 3rd International Congress of Endocrinology 3. Mexico City, Excerpta Medica, 220, 1969. 27. Schein, P. S., and Bates, R. W.: Plasma glucose levels in normal and adrenalectomised mice treated with streptozotocin and nicotainamide. Diabetes 17:760, 1968. 28. Stauffacher, W., Burr, I., Gutzeit, A., Beaven, D., Veleminsky, J., and Renold, A. E.: Streptozotocin diabetes: time course of irreversible P-cell damage: further observations on prevention by nicotinamide. Proc. Sot. Exp. Biol. Med. 133:194, 1970. 29. Dulin, W. E., and Wyse, 8. M.: Studies on the ability of compounds to block the diabetogenic activity of streptozotocin. Diabetes 18 ~4.59, 1969.
J. Pearson,
R. G. Larkins,
Total plasma immunoreactive insulin (1RI) and percentage “big” insulin (proinsulinlike material) were measured in a patient with an inoperable islet cell carcinoma who did not improve clinically after streptozotocin administration. Prior to the streptozotocin, the total IRI rose with administration of tolbutamide, and the percentage of “big” insulin remained constant. After intravenous glucose there was little change in plasma IRI, but a substantial rise in the percentage of “big” insulin occurred. The admin-
and F. I. R. Martin
istration of streptozotocin was acutely with hypoglycemia, a associated slight increase in total IRI, and no change in the percentage “big” insulin. With chronic administration and extension of the disease, the total IRI and percentage “big” insulin rose progressively. It is concluded that the percentage “big” insulin rises as loss of tumor differentiation occurs and that changes in the percentage of “big” insulin after streptozotocin may give an early indication of likely response to streptozotocin.
S
TREPTOZOTOCIN is an antibiotic isolated from Streptomyces achromogenes1~2 that has cytotoxic properties specific for the P-cells of the pancreas.3-5 Its use in the treatment of several patients with metastatic islet cell carcinoma has been reported, with excellent symptomatic relief and regression of the tumor in some instances 6-11 but only partial benefit in others.12s13 Blackard et aL7 investigated qualitative aspects of plasma insulin in a patient following successful treatment with streptozotocin. They found that the high proportion of “big” insulin (proinsulinlike material) seen initially after oral glucose was reduced after therapy, despite minimal change in plasma immunoreactive insulin (IRI) concentrations. In contrast, the present report describes changes in “big“ insulin and IRI in a patient who did not respond to streptozotocin. It is hoped that some predictive factors regarding potential response to streptozotocin may emerge from reports such as this. CASE REPORT A 56-yr-old man presented to another hospital with a 2-mo history of abdominal pain, anorexia, weight loss, and diarrhea. The symptoms bore no relationship to meals or fasting. The only symptom directly attributable to hypoglycemia was a recent preference for sweet foods for breakfast. The liver was hard and irregular and extended 8 cm below the costal margin.
From the Department of Biochemistry and Endocrinology, The Royal Melbourne Hospital, Melbourne, Auslralia. Received for publication October 15, 1971. Margaret J. Pearson, M.Sc.: Endocrine Biochemist, Department of Biochemistry, Royal Melbourne Hospital, Melbourne, AustraZia. R. G. Larkins, M.B.B.S., M.R.A.C.P.: A&slant Endocrinologist, Royal Melbourne Hospital, and Associate, University of Melbourne Department of Medicine, Royal Melbourne Hospital, Melbourne, Australia. F. I. R. Martin, M.D., F.R.A.C.P. : Endocrinologist, Royal Melbourne Hospital, and Senior Associate, University of Melbourne Department of Medicine, Royal Melbourne Hospital, Melbourne, Australia. Metabolism,
Vol. 21, No. 6 (June), 1972
661
552 Laparotomy
PEARSON,
revealed
a large
tumor
mass
LARKINS,
in the body of the pancreas,
AND MARTIN
with
multiple
hepatic metastases and a distended gall bladder. A prophylactic cholecvstoieiimostomv was performed. Six hours postoperatively, after awaking from the anesthetic, the patient lansed into a deep coma. Blood sugar at that time was 12 mg/lOO ml. and the patient responded promptly to intravenous dextrose. Histologic examination of the operative biopsy revealed cords and clumps of cells, varied in size and shape, often with large and dark nuclei and numerous mitoses, consistent with an islet cell carcinoma of the pancreas. Plasma IRI levels ranging between 78 uU/ml and 142 pUlm1 in the presence of hypoglycemia confirmed this diagnosis. Therapy with corticosteroids (dexamethasone 16 mg daily) and diazoxide (800 mg daily) was ineffective in controlling hypoglvcemia, and a continuous intravenous infusion of 20% fructose was required. This resulted in the development of lactic acidosis fplasma lactate 46 mg/lOO ml, normal 5-15 mgboo ml and serum bicarbonate 7 meq/liter, normal 20-30 meq/ liter), so that 10% dextrose was substituted. Three weeks postoperatively a combination of glucagon (8 mg infused intravenously daily), dexamethasone (12 mg infused intravenously daily), and diazoxide (100 mg orally at 6-hr intervals) provided partial control. However, when intravenous fluids and glucagon injections were discontinued occasional severe hypoglycemic episodes recurred, despite the maximum tolerated doses of diazoxide and dexamethasone. Peripheral edema developed, and bendroflumethiazide, .S mg daily, was added. In view of the failure to adequately rontrol the hypoglycemia, streptozotocin (Upjohn International Inc.) was administered s wk postoperatively. Two doses of 1.5 g, made up in citrate buffer pH 4.4 immediately before injection, were given intravenously, with 7 days between doses. Each dose was followed by nausea and vomiting, but not by hypoglycemia. For 2 wk there was symptomatic improvement, but then hypoglycemia recurred. A further 3 g of streptozotocin i.v. resulted in severe hypoglycemic episodes immediately and z hr and 12 hr later, in spite of continuous infusion of fructose. After 2 wk more in which the patient was comparatively well the symptoms recurred, and the liver had obviously increased in size. A further 1.5 g of streptozotocin was given intravenously, and 2 days later 1.5 g was infused into the celiac artery. Intravenous fructose was again required to control acute hypoglycemia. Pulmonary and periphera1 edema developed that was unresponsive to diuretic therapy, digoxin, and aminophylline, and the patient died from pulmonary edema 28 hr after the last dose of streptozotocin. Liver function tests throughout the patient’s illness were abnormal with markedly elevated glutamic-oxaloacetic transaminase and alkaline phosphatase; no further change occurred after streptozotocin. Liver scan 1 mo postoperatively, prior to the streptozotocin, showed evidence of hepatic metastases, and 2 mo later, just prior to death, a repeat scan showed further growth of the metastatic lesions. Blood urea was normal and did not rise following streptozotocin. There was no evidence of renal tubular dysfunction. Serum potassium fell to 3.5 meq/liter (normal 3.5-5.5 meq/liter) whiIe the patient was on diuretic therapy. The hemoglobin was slightly reduced initially but did not fall following streptozotocin. Serum calcium and immunoassayable gastrin were normal, and s-hydroxyindoleacetic acid was not detected in the urine. Autopsy revealed a 3 cm diameter white necrotic tumor in the head of the pancreas. Metastatic tumor was present in portal lymph nodes and in the massively enlarged liver. Gross postmortem autolysis made estimation of the degree of necrosis in the tumor impossible.
MATERIALS
AND METHODS
Blood sugar, plasma IRI, and the percentage of “big” insulin (measured as immunoreactive insulin) were determined during both an intravenous glucose tolerance test 7.50 mg/kg) and in intravenous tolbutamide tolerance test (1 g) performed on successive days 1 wk prior to streptozotocin administration. Neither test was performed under basal conditions, as hypoglycemic episodes prevented this. An infusion of 5% dextrose at a constant rate of 1 liter/l2 hr was continued up to 1 hr before the time of the glucose injection in
TREATMENT OF CELL CARCINOMA WITH STREPTOZOTOCIN
5(
-
INSULIN.
.__
PRO-INSULIN
3c
Fig. 1. Solid line represents standard curve obtained by displacement of 126l-labeled insulin from antibody by unlabeled insulin. Solid dots show points obtained by serial dilution of freeze-dried extracts of “little” insulin fraction obtained (see text). Interrupted line represents standard curve obtained for proinsulin, and partially open dots show points obtained by serial dilution of freeze-dried extracts of “big” insulin fraction obtained.
20
10
o.; COLD
STANDARD
ADDED
hglml)
Blood sugars were measured by the Autoanalyser (Technicon) ferricyanide method,14 and plasma IRI by the charcoal method of Herbert et al.15 as modified by Pearson et al,16 using 125-I insulin and a pork insulin standard (Lilly, Lot P.J. 5589). Insulin sensitivity (KIw) was determined as previously described.17 Plasma “big” and “little” insulin were assessed by a modification of the method of Roth et al.,18 as described by Blackard et al.,’ except that the buffer used was 0.01 M Verona1 the glucose tolerance test, and the same infusion was continued at a constant rate throughout the tolbutamide tolerance test. The plasma IRI and percentage “big” insulin were monitored acutely and chronically following the administration of streptozotocin. The values were never obtained under truly basal conditions due to the necessity of frequent (2-hr intervals) glucose feeding during the night prior to the tests.
PRO-INSULIN 0
FRACTION NUMBER
INSULIN
Fig. 2. Elution pattern of porcine proinsulin and porcine insulin standard preparations on Sephadex G-50 column.
554
I.R.I.
-uhl
PEARSON,
LARKINS.
AND MARTIN
as. Xet h&
II
I
TIME AFTER-Ii.
GLUCOSi
(MN)
A
TIME
AFTER
t.v.
TOLBUTAMlOE
Fig. 3. (A) Blood sugar (B.S.) shown by continuous line, total I.R.I. shown by gray columns, and percentage “big” insulin (B.I.) shown by black columns, after intravenous administration of 750 mg/kg glucose i.v. (B) Blood sugar, total I.R.I., and percentage “big” insulin after 1 g tolbutamide i.v. Symbols are same as in Fig. 3A.
(mid
B
buffer pH 7.4 containing 0.2% sodium azide and that 2 ml of plasma was used for each sample. “Big” insulin isolated in this way is probably a crude preparation of proinsulin. In preliminary experiments, “big” insulin and “little” insulin obtaned from plasma by this method were characterized by comparison with standard porcine proinsulin (Lilly, Lot 6151082&46.2) and insulin. The immunologic behavior on serial dilution of freeze-dried fractions (Fig. 1) and the elution pattern on Sephadex G-50 (Fig. ?-) confirmed the differing molecular weights and immunoreactivity of “big” and “little” insulin. In the subsequent text, percentage “big” insulin refers to the insulin eluting as “big” insulin on Sephadex G-50 read from the insulin standard curve expressed as a percentage of the total irnmunoreactive insulin in the whole serum, again read from the insulin standard curve.
RESULTS Intravenous glucose produced only a small increase in plasma insulin, although the percentage of “big” insulin increased from 14% at fasting to 45% 4.5 min after the glucose injection (Fig. 3A). In contrast, intravenous tolbutamide produced a marked increase in plasma IRI accompanied by a prolonged and exaggerated fall in blood sugar, occurring after an initial unexplained rise. The percentage contribution of “big” insulin to the total IRI after tolbutamide did not change significantly (Fig. 3B), reflecting a considerable output of “big” insulin as well as “little” insulin. However, the percentage “big” insulin at the commencement of this test was considerably higher than the initial level during the glucose tolerance test on the previous day, perhaps because of the continuous slow glucose infusion.
TREATMENT
OF CELL CARCINOMA
A
Fig. 4. (A) Blood sugar (B.S.) (continuous line), total I.R.I. (gray columns), and percentage “big” insulin (B.I.) (black columns) in 4 hr after single dose of 1.5 g streptozotocin intravenously, given at time indicated by arrow. (B) Total I.R.I. (gray columns) and percentage “big” insulin (B.I.) (black columns) over 2 mo. during which streptozotocin was given. Arrows represent doses of streptozotocin.
B
WITH STREPTOZOTOCIN
555
TIME
TIME
Initially, insulin sensitivity was less than normal (KITT 1.2, normal > 2.4), and after 6 g of streptozotocin had been given this remained unchanged. Administration of a total of 9 g of streptozotocin over an 8-wk period did not appear to significantly affect either plasma IRI or the percentage “big” insulin. The levels of both increased in parallel with clinical progression of the tumor, the plasma IRI rising to over 500 rU/ml, and the percentage “big” insulin reaching 80% (Fig. 4B). All readings were obtained under similar conditions, with 2-hr glucose feeds throughout the previous night up to 2 hr before the test was taken. After the second test shown on Fig. 4B, the patient was also receiving 400 mg diazoxide daily, 6-8 mg dexamethasone daily, and 5 mg bendroflumethiazide daily. These agents should have decreased the plasma insulin, but a progressive rise occurred in spite of them. Similarly, streptozotocin appeared to have little acute effect on either the plasma IRI or “big” insulin over a 5-hr period immediately following the intravenous injection of 1.5 g streptozotocin (Fig. 4A). Although the blood sugar dropped precipitously to 10 mg/100 ml 2% hr after the injection, there was only a slight increase in the plasma IRI (460-510 pU/ml) and no significant change in the percentage “big” insulin (62-60%) at the time of maximal hypoglycemia.
PEARSON,
556
LARKINS,
AND MARTIN
DISCUSSION
This patient with a metastatic islet cell carcinoma showed high plasma IRI levels with a proportion of “big” insulin that increased from normal to extremely high levels during the course of the disease. These findings are compatible with previous reports, both of norma120s21 and high21-25 levels of proinsulin or proinsulinlike material in patients with islet cell tumors. The tumor showed a varied pattern of response to stimuli. The proportion of “big” insulin increased following glucose, as has been reported for normal subjects,lg but remained virtually unchanged after tolbutamide. However, this represented a large increase in the absolute quantity of “big” insulin when the rise in total IRI was taken into account. Gutman et al. found in a patient with a benign insulinoma that tolbutamide apparently released “big” insulin with 71% of the plasma IRI in the proinsulinlike fraction after tolbutamide and only 5% after oral glucose. 21 In contrast, Gorden and Roth reported a patient with an islet cell carcinoma in whom the percentage of proinsulinlike material fell from 37 to 3% 15 min after intravenous toIbutamide.25 Similarly, the patient of Melani et al. with a benign tumor showed a fall in the proportion of proinsulin following tolbutamide,24 and there was little change in the absolute proinsulin concentration following tolbutamide in the patient of Taylor et a1.,8 who like our patient had a malignant lesion. In contrast to the majority of reported cases, this patient apparently did not respond to streptozotocin. The dose given was similar to that used by the majority of workers, 6,7*11 but much less than the total dose of 30 g given by Stanley et a1.g The characteristic hypoglycemic episodes following the administration of the drug confirmed its activity. Stanley et al9 suggested that injection into the cehac artery was the most effective route, although this increases the danger of nephrotoxicity due to inadvertent administration into the renal artery. The last dose given to this patient was injected into the celiac artery, but survival after this was too short to assess either its efficacy or side effects. The refractory pulmonary edema that caused the patient’s death was probably due to the combination of intravenous fluid, diazoxide, and corticosteroids, although a possible role of streptozotocin could not be discounted. The relative ineffectiveness of streptozotocin in the present case may have been due to the massive size of the tumor or to the particular abnormality of the enzymic constitution of the tumor cells. The mechanism of the P-cytotoxic effect of the drug is not known. TayIor 26 demonstrated a direct inhibitory effect on insulin synthesis, but in practice a reduction in plasma insulin is not always found even in patients who respond symptomatically to the drug.’ It has also been postulated27‘2s that streptozotocin interferes with nicotinamide adenine dinucleotide formation in the P-cell, but it is not known whether an alteration in P-cell pyridine nucleotide content would result in diabetogenecity. After acute administration of streptozotocin in the present case, there was no striking alteration in the plasma insulin concentration or in the percentage of “big” insulin; in spite of this, marked hypoglycemia occurred (Fig. 4A).
TREATMENT
OF CELL CARCINOMA
WITH STREPTOZOTOCIN
557
This may have been due to altered peripheral sensitivity to insulin or to change in biologic but not immunologic reactivity of the “little” insulin released. Alternatively, the slight increase in total IRI may have accounted for the hypoglycemia. Altered sensitivity to insulin after streptozotocin appears unlikely, as there was no significant change in KI.L.~. after chronic administration. To our knowledge, the only other published study reporting changes in proinsulin after streptozotocin is that of Blackard et al7 In their patient, clinical improvement coincided with a fall in percentage of “big” insulin found after oral glucose, although no information on fasting levels was given. It is likely that the increase in the percentage “big” insulin we observed was a manifestation of progression of the disease with further loss of differentiation of the tumor cells. This supports the suggestion7 that the proportion of “big” insulin may be a marker reflecting the abnormality in insulin biosynthesis and release. This is in accord with the observation of Gutman et a1.21 that the highest percentage of proinsulinlike material occurs in malignant islet cell tumors. The rise in percentage “big” insulin occurring despite streptozotocin may be an early indication of insensitivity to this agent. It is hoped that further studies of IRI and proinsulinlike material in plasma, together with their acute response to stimuli such as tolbutamide and glucose, may define the group of patients with inoperable islet cell carcinomas most likely to respond to streptozotocin. ACKNOWLEDGMENT The help of Dr. B. King, who initially referred the patient, and Dr. L. Murton, who assisted in obtaining the clinical data, is gratefully acknowledged. The streptozotocin was supplied by Upjohn International Inc., and the proinsulin by Eli LiIly. Dr. J. Hansky of the Department of Medicine, Monash University, performed the gastrin assay.
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B. M., Bennett, A., Edwards, J. C., and Taylor, K. W.: Treatment of multiplehormone producing malignant islet-cell tumour with streptozotocin. Lancet 2 :895, 1968. 7. Blackard, W. G., Garcia, A. R., and Brown, C. L.: Effect of streptozotocin on qualitative aspects of plasma insulin in a patient with a malignant islet cell tumour. J. Clin. Endocr. 31:215, 1970. 8. Taylor, S. G., Schwartz, T. B., Zannini, T. J., and Ryan, W. G.: Streptozotocin therapy for metastatic insulinoma. Arch. Intern. Med. (Chicago) 126:654, 1970. 9. Stanley, N. N., Marks, V., Kreel, L., and McIntyre, N.: Streptozotocin treatment of malignant islet cell tumour. Brit. Med. J. 3:562,1970. 10. Arnould, Y., Ooms, H. A., and Bastenie, I’. A.: Treatment of insulinoma with streptozotocin. Lancet 1:1210, 1969.
558 11. Sadoff, L.: Effects of streptozotocin in a patient with islet cell carcinoma. Diabetes 18:675,1969. 12. Livingston, R. B., and Carter, S. K.: Chemotherapy Fact Sheet, Program Analysis Branch, Chemotherapy, National Cancer Institute, Bethesda, Md. 1969, p 1. 13. Ennis, G.: Personal communication. 14. Hoffman, W. A.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:.51, 1937. 15. Herbert, V., Lau, K., Gottlieb, C. W., and Bleicher, S. J. : Coated charcoal immunoassay of insulin. J. Clin. Endocr. 25:1375, 1965. 16. Pearson, M. J., Fullerton, M. J., Martin, F. I. R., and Melick, R.: An assessment of the charcoal immunoassay of insulin. Proc. Aust. Ass. Clin. Biochem. 1:315, 1968. 17. Martin, F. I. R., Pearson, M. J., and Stocks, A. E.: Glucose tolerance and insulin insensitivity. Lancet 1:1285, 1968. 18. Roth, J. Gorden, P., and Pastan, I.: “Big insulin”: a new component of plasma insulin detected by immunoassay. Proc. Nat. Acad. Sci. USA 61:138, 1968. 19. Melani, F., Rubenstein, A. H., and Steiner, D. F.: Human serum proinsulin. J. Clin. Invest. 49:497, 1970. 20. Gutman, R. A., Lazarus, N. R., Penhos, J. C., Recant, L., and Fajans, S. S.: Proinsulin (P.I.) and proinsulin-like material (PI-LM) in serum of patients with islet cell tumours. Diabetes X3:360, 1969. 21. -, -, -, Fajans, S., and Recant, L.: Circulating proinsulin-like material in func-
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tioning insulinomas. New Eng. J. Med. 284: 1003,197l. 22. Lazarus, N. R., Tanese, T., Gutman, I’. A., and Recant, L.: Synthesis and release of proinsulin and insulin by human insulinoma tissue. J. Clin. Endocr. 30:273, 1970. 23. Goldsmith, S. J., Yalow, R. S., and Berson, S. A.: Significance of human plasma insulin sephadex fractions. Diabetes 18:834, 1969. 24. Melani, F., Ryan, W. G., Rubinstein, A. H., and Steiner, D. F.: Proinsulin secretion by a pancreatic beta-cell adenoma. New Eng. J. Med. 83713, 1970. 25. Gorden, P., and Roth, J.: Plasma insulin: fluctuations in the “big” insulin component in man after glucose and other stimuli. J. Clin. Invest. 48:2225, 1969. 26. Taylor, K. W.: Regulation of insulin synthesis. In 3rd International Congress of Endocrinology 3. Mexico City, Excerpta Medica, 220, 1969. 27. Schein, P. S., and Bates, R. W.: Plasma glucose levels in normal and adrenalectomised mice treated with streptozotocin and nicotainamide. Diabetes 17:760, 1968. 28. Stauffacher, W., Burr, I., Gutzeit, A., Beaven, D., Veleminsky, J., and Renold, A. E.: Streptozotocin diabetes: time course of irreversible P-cell damage: further observations on prevention by nicotinamide. Proc. Sot. Exp. Biol. Med. 133:194, 1970. 29. Dulin, W. E., and Wyse, 8. M.: Studies on the ability of compounds to block the diabetogenic activity of streptozotocin. Diabetes 18 ~4.59, 1969.