Glutathione and glutathione linked enzymes in human small cell lung cancer cell lines

CANCER LETTERS Cancer

Glutathione

Letters

75 (1993) I I I-l I9

and glutathione linked enzymes in human small cell lung cancer cell lines

Rashmi Sharmaa, Sharad S. Singhalb, Sanjay K. Srivastavaa, Krishna K. Bajpai”, Eugene P. Frenkel’, Sanjay Awasthi*b ‘Department

of Human

Biological

Chemistry

Galveston, h Department University

‘Division of

of Internal

qf

Hematology

Texas

and Genetics,

Medicine,

Medical

and Oncology.

I5 May 1993; revision

Galveston.

University of Texas TX 75235,

received

ofTexas

Medical

Branch.

USA

Division of Hematology

Branch,

Dallas.

(Received

University

TX 77555-1067.

and Oncology.

TX 77555-0565. Southwestern

USA Medical

Center.

USA

28 September

1993; accepted

29 September

1993)

Abstract Glutathione levels and several glutathione-linked enzyme activities have been variably correlated with cisplatin chemosensitivity in cultured neoplastic cells, In order to determine the relative contribution of the glutathione-linked enzymes towards mediating inherent cisplatin resistance in cancer cells, we have measured the chemosensitivity to cisplatin, glutathione levels and activities of glutathione S-transferase, glutathione peroxidase. glutathione reductase and glucose-6-phosphate dehydrogenase in 8 cultured human small cell lung cancer (SCLC) cell lines with widely differing cisplatin sensitivities. Of these parameters, only glutathione S-transferase activity correlated with degree of cisplatin

resistance in a linear fashion. Key

words;

Cisplatin;

Small cell lung cancer;

Drug resistance;

1. Introduction Cisplatin (cis-diamminedichloroplatinum (II), DDP) is one of the most effective antineoplastic drugs for the treatment of human small cell lung cancer, but its efficacy is limited because malignant cells often display de novo or acquired resistance to its cytotoxic effects [ 1.21. The cytotoxic effects of DDP are related to its ability to form * Corresponding 0304-3835/93/$06.00 SSDI

author. 0

0304-3835(93)03196-C

1993 Elsevier

Scientific

Publishers

Ireland

Glutathione:

Glutathione

S-transferase

platinum coordinates with cellular nucleophiles (such as DNA) through electrophilic attack and through a variety of redox mechanisms [3]. Glutathione (GSH), the predominant acid-soluble thiol in cells is the main defense against the toxicity of electrophilic and redox-active compounds such as DDP. Formation of GSH-electrophile thioether conjugates spontaneously or through reactions catalyzed by glutathione S-transferases (GSTs) and the subsequent formation of mercapturic acids from these conjugates is a major pathway for Ltd. All rights reserved.

112

detoxification of electrophiles [4]. DDP is known to form conjugates with GSH [5]. DDP has also been shown to result in the formation of lipid hydroperoxides [3] which are substrates for cellular glutathione peroxidases (GPx), cellular enzymes which couple the oxidation of GSH with the reduction of reactive peroxides. The resultant oxidized glutathione-disulfide (GSSG) is subsequently reduced to GSH by glutathione-reductase (GR) which couples the reduction of GSSG with the oxidation of NADPH [6]. Glucose-6-phosphate dehydrogenase (6-6-PD), the rate limiting enzyme of the pentose phosphate shunt is largely responsible for maintaining NADPH in the reduced state [7]. The elements of this interlinked system of defense against the toxic effects of electrophilic/oxidant toxins have been shown alone and in combinations to be variably increased in DDPresistant cancer cells in vitro in several studies [8-281 which have compared these parameters between a sensitive parental cell line and a derived DDP-resistant cell line. In the present studies we have attempted to determine whether the DDPsensitivity (ICsO) of eight small cell lung cancer (SCLC) cell lines from different patients correlates better with GSH levels or with the activities of GST, GR, GPx or G-6-PD. The results of our studies indicate that GSH levels, and activities of GR, GPx and G-6-PD did not correlate with their IC,,for DDP. In contrast, GST activities were correlated in a linear fashion with DDP-resistance, nd that the predominant GST isoenzyme in all these cell line was a *-class GST (pl 4.9; M’ = 22 500). The significance of our findings are discussed in relation to previous studies which have investigated the link between DDP-resistance and the components of the GSH-linked electrophilic/oxidant defense system.

chased from Sigma Chemicals Co., St. Louis. MO. cis-diamminedichloroplatinum (II) was obtained from Bristol Laboratories, Evansville, IN. Cell culture supplies including fetal bovine serum (FBS), RPMI- 1640 medium, and penicillin/streptomycin/ amphotericin solution (PSA) were purchased from Gibco Laboraties, Grand Island, NY. 2.2. Cell lines All eight human small cell lung cancer cell lines, NC1 H-69, H-82, H- 128, H- 187. H-524, H-774, H1173 and H-2496 were generously donated by Dr. Herbert Oie at the National Cancer Institute, Baltimore, MD. These cell lines were grown in RPMI-1640 medium containing 10% FBS and 1% PSA at 37°C in a 5% CO1 atmosphere in a Forma Scientific water jacket incubator. Cells were maintained in log phase of growth by passaging them in 1:3 dilution with medium every 3-4 days. L 3. DDP chemosensitivit? The number of viable cells after cisplatin treatment was estimated using an MTT cytotoxicity assay modified from that previously described by Carmichael et al. [29]. Cells in log phase were diluted 1:3 with medium. Aliquots of these cells were inocculated into 96-well plates to which DDP (concentrations ranging from 1 to 80 PM) was added. Cells were allowed to grow for 7 days in presence of cisplatin. After 7 days, l-ml aliquots of these cells were incubated with 30 ~1 MTT (2 mgiml) for 1 h at 37°C. The medium was then removed by centrifugation and the pellet solubilized using 1 ml isopropanol containing 0.04 N HCl. Absorption was recorded at 560 nm using a gilford response spectrophotometer against a blank consisting of cells without MTT, solubilized in acid isopropanol.

2. Materials and methods 2.1. Chemicals and reagents 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB), lchloro-2,4_dinitrobenzene (CDNB), P-nicotinamide adenine dinucleotide phosphate (NADP). NADPH, D-glucose-6-phosphate (G-6-P), flavin adenine dinucleotide (FAD). cumene hydroperoxide (CuOOH) and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) were pur-

2.4. Measurement of GSH and erq~mr activities Fully grown cells ( 100 ml) in log phase were centrifuged at 150 x g in a Sorvall RCSC centrifuge. The resultant cell pellet was washed twice with 50 ml phosphate-buffered saline, subjected to lysis in 1 ml hypotonic buffer (10 mM potassium phosphate, pH 7.0) and homogenized by sonication on ice at 50 W for 15 s four times in Sonilier Cell Disruptor. The homogenate was immediately spun

R. Sharma et al. /Cancer Lett. 75 (19931 111-119

at 16 000 x g for 20 min and the supernatant collected for further analyses. After removing aliquots for protein determination by Bradford’s method [30] and GSH determination, P-mercaptoethanol was added to homogenate to achieve a final concentration of 1.4 mM. Glutathione was measured using an enzymatic-HPLC method previously reported by us [31]. This method is based on using HPLC to quantify the GSH-CDNB conjugate formed in cell homogenate in presence of excess GST and is highly specific for. GSH. GST activity towards CDNB as substrate was determined using the method of Habig et al. [32]. GR activity was determined using Beutler’s method [33]. GPx activity using cumene hydroperoxide as substrate was determined by the method of Awasthi et al. [34]. G-6-PD activity was determined by the method of Glock and McLean [35]. Values for GSH and enzymatic activities were expressed as mean + S.D. from three identical sets of experiments. 2.5. Purifi;cation and characterkation qf GSTs Purification and characterization of GSTs were performed as previously described [36]. Briefly, supernatant of cell homogenate prepared as described above was incubated with 2 ml GSH-linked epoxy-activated Sepharose 6B resin at 4°C overnight on a shaker. The resin mixed with supernatant was centrifuged and washed with 22 mM potassium phosphate buffer (pH 7.0) containing 1.4 mM /3-mercaptoethanol to remove unbound proteins. The adsorbed enzyme was then eluted with 50 mM Tris-HCl (pH 9.6) containing 1.4 mM /3-mercaptoethanol and 10 mM GSH. All the procedures were carried out at 4°C. After dialysis, the enzyme was subjected to column isoelectric focusing as previously described [36]. The GST activity towards CDNB was determined in eluted fractions. Purified total GSTs were used for SDSPAGE, isoelectric focusing and Western blot analyses using previously described antibodies specific for the human CY,p and r classes of isoenzymes. 3. Results and discussion The SCLC cell lines used in these studies grew as clumps suspended in medium. The MTT-assay

113

for quantifying viable cells was standardized by comparing absorption at 560 nm of the acid isopropanol extract of formazan-stained cells with the number of trypan blue excluding cells in a suspension of trypsin-disaggregated cells counted in a hemocytometer. The range of linearity of the MTT assay was thus determined to be between 2 x lo5 to 2 x lo6 cells per ml medium (data not presented). DDP sensitivity assays were performed in three separate experiments and the inter-experimental variation of IC,” was about 10%. The DDP sensitivity curves shown in Fig. 1 represent an average of three determinations for each of the cell lines. The ICso for DDP determined from these curves are given in Table 1. GSH content and activities of GSH-related enzymes are presented in Table 1. GSH levels in the cell lines examined were similar to those previously reported for similar cell lines [ 10,24,26,37.38]. We observed a greater inter-experimental variation in determination of GSH content than in GSHlinked enzyme activities for each cell line. This may be attributable to minor variations in nutritional conditions in the medium since GSH content is known to vary with changes in nutritional conditions and cell density in culture [39], Although GSH content varied between different cell lines, there was no correlation between the I& for DDP and GSH content (r’ = 0.05). GR and GPx activities were found to be within the range of those previously reported for similar cell lines [38]. The activities of GR and GPx were more variable between different cell lines than GSH content, but again there was no correlation between their activities and ICsO for DDP (Y’ = 0.459 and 0.056, respectively). The G-6-PD activities in our SCLC cell lines were in the range previously reported for GLC,-SCLC cells [40]. The variation in G-6-PD activity may be partially attributable to the normal inter-individual variation in G-6-PD. No correlation was found between G-6-PD and IC,, of DDP (v’ = 0.212). Of the parameters measured, the widest variation between cell lines was seen in GST activity. A close correlation between GST activity and the ICso for DDP was observed (r7 = 0.987) (Fig. 2). The most resistant cell line (H-69) contained 34-fold higher GST activity than the most sensitive cell line (H-2496). Purification and characterization of GSTs from each

114

R. Sharntu

CI crl. /Cuncrr

Lrtr

75 (1993)

Ill-II9

25

0

20

40

60

80

Cisplatin (PM) Fig. I. Chemosensitivity of SCLC cell lines towards DDP. Percentage of viable cells determined by MTT-assay are plotted against concentration of DDP to which cells were exposed. Each point represents an average of three separate determinations. ICs,, for each cell line was determined from this plot.

Table 1 Quantitation

of GSHh and GSH-related

enzymeb

Cell line

Chemosensitivity DDP-IC,, (PM)

GSH (nmolimg protein)

NCIIH69 NCI-H82 NCI-HI28 NCI-HI87 NCI-H524 NCI-H774 NCI-HI 173 NCI-H2496

36.6 25.5 14.5 6.8 5.0 3.5 6.8 4.4

63.9 61.2 51.0 52.7 48.9 70.6 75.2 37.6

dn = 6. %i = 3. ‘One mU of enzyme activity

was defined

+ + * + f + f f

as

9.1 6.4 4.4 6.8 6.2 4.7 7.9 8.9

activitiesC m certain

I nmol substrate

small lung cancer CR ImUimg protein)

GST (mU/mg protein) 316.95 232.42 160.0 36.99 15.0 15.85 53.93 9.3

human

f + f zt

6.3 1.4 20.0 2.0 l I.0 * I.1 f 3.6 f 0.5

consumed

52.6 62.23 15.0 12.97 43.7 26.00 31.56 IX.0

per min

f f * * f f zt f

cell lines G-6-PD mU/mg protein)

GPX (mU/mg protein) 0.4 1.3 0.8 0.5 0.2 2.4 2.4 2.0

3.x 33.80 1.3x 10.81 21.36 20.35 45.47 9.87

f * f + f * * f

0.7 4.3 0.1 0.7 6.1 2.‘) 3.‘) 0.8

-_-. ‘3’ 60 17.56 102.20 112.02 71.07 49.28 --_. 11’04 N.D.

zt 2.8 f 0.4 l 15.7 * 2.9 * 4 5 f 0.7 f 7.2

*GSH 320

n GST

*GR JZGPx iG6PD

20 IC-50 for DDP Fig. 2. The relationship of GSH content and activites of GST. GPx, CR. and G6PD to the DDP-IC,, of eight human SCLC cell lines. The 1C,, for DDP of each cell line is plotted against GSH content (nmolimg protein) and activity of each enzyme (mU/mg protein). Linear regression was used to draw the best lit line for each parameter,

of the cell lines revealed that an anionic GST (pl 4.9) was the only isoenzyme observed in all the cell lines, consistent with previous reports [22,26]. A representative IEF profile of GSTs from the H-69 cell line demonstrating the pl 4.9 isoenzyme is shown in Fig. 3. The SDS-polyacrylamide gel electrophoresis (Fig. 3, inset A) of the purified protein from H-69 cells showed a single protein band at Mr. 22.500. A Western blot using polyclonal antibodies specific for the CY.p and 7rclasses of GSTs (Fig. 4) revealed that the GST isoenzyme from H-69 cells were recognized only by anti-GST x antibodies. Similar results were observed with the other seven cell lines (data not presented).

The results of present studies indicated that among the elements of the GSH-linked detoxilication studied, GST activity was a more important determinant of inherent DDP-resistance than either GSH content or activities of CR, GPx or G6-PD in our human SCLC cell lines (not previously exposed to incremental doses of DDP in vitro). GSH values were similar for all cell lines. The lack of correlation between GR, GPx and G6-PD activity with DDP-resistance suggests that the GSH-linked lipid peroxidation defenses are not limiting factors in defending against DDPcytotoxicity in these cell lines under present conditions. Similarly, the lack of correlation between GSH content and DDP-resistance implies that in

116

R. Sharma et al. /Cb~crr

Lett. 75 (1993)

III-119

pI=4.9

0.16

f

k

“SC;

0.12

x

B $ Y G w

x X

A

‘X

94K -

0.08

x x

43K

-

20.1K

-

,

0.04

0

0

20

40

60

80

100

Fraction Number Fig. 3. GST isozymes of the H-69 cell line. The IEF profile of GST activity in the affinity purified enzyme showed one peak at The SDS-PAGE (inset A) revealed the presence of one protein band at M, __ ‘7 500. GST activity (0) and pH (xl.

these cell lines under present in vitro conditions, GSH concentration was also not a limiting factor in defending against DDP-cytotoxicity. In this respect, our results are in agreement with a previous similarly designed study by Armstrong et al. [41] showing that in six different human breast cancer cell lines not previously exposed to chemotherapeutic drugs in vitro, GST activity correlated with resistance to Hepsulfam (an alkylating agent), whereas GSH content did not. A possible explanation for the lack of correlation of DDP-resistance with GSH level may be that under present culture conditions, GSH synthesis in all cells is at a maximum and determined primarily by culture conditions rather than intrinsic differences in the ability of the cells to synthesize GSH. Furthermore, DDP concentration (in the micromolar range) to which cells were exposed were several fold lower than the concentrations of GSH in these cells (approx-

pl = 4.9.

imately 2-3 mM). Thus, a slight increase in GSH concentration may not significantly impact the rate of conjugation of DDP with GSH. In contrast to the result of Armstong’s studies [41], a study with similar design to ours by Mistry et al. [42], showed that DDP resistance was better correlated with GSH levels than GST activity. A possible explanation for the differences may be either inherent differences in resistance mechanisms which can be expressed by cells of differing histologic types or differences in the methodology of cell-culture or biochemical determinations. It should be noted that in other studies of human cell line of several histologic types which have compared GSH-linked parameters between a parental cell line and in vitro-derived resistant cell lines, the most common GSH-linked parameters correlated with DDP resistance observed were either GSH level or GST activity or both [8-281. Perhaps more

R. Sharma

et al. /Cancer

Lerr. 75 (1993)

117

111-119

25K

26.5K

-

1

Anti T

Anti p

Anti CY

22.5~

~mm

2

1

interesting is the fact that in those DDP-resistant cell lines in which GSH levels were not elevated GST activity was found to be increased [9,20,21, 261 and in those DDP-resistant cell lines in which GSH was elevated, GST activity did not correlate with DDP-resistance [21,24,27]. This observation points towards the importance of the GSH-electrophile detoxification mechanism in mediating DDP resistance and suggests that an enhanced rate of conjugation of GSH with DDP in cells may be achievable by either increasing the concentration of the substrate (GSH) or the catalyst (GST). In this respect, it should be noted that the nonenzymatic conjugation of DDP (a hydrophilic compound) with GSH has never been shown to be catalyzed by GSTs (enzymes with affinity for hydrophobic-electrophilic substrates) under invitro assay conditions. Thus, it is difficult to understand why DDP-resistance in our and other studies has been correlated with increased GST. It is possible that this is an artifact of culture conditions. Another intriguing explanation may be rooted in the fact that several GSH-electrophile conjugates are known to inhibit the activity of GSTs [43]. Thus, if the non-enzymatically formed GSH-DDP conjugate is an inhibitor of GSTs, cells with greater GST activity may be at an advantage in their ability to conjugate other exogenous

1

2

Fig. 4. Western blot analysis of human SCLC cell line H-69. Western blots of GSH affinity bodies specific for (Y, p, and r-class GSTs showed that the protein was recognized

+%:

2

purified total GSTs using polyclonal only by the anti-GST-n antibodies.

anti-

(cellular waste products which accumulate in medium) or endogenous toxic hydrophobic electrophiles. A better understanding of the kinetics of the DDP-GSH conjugate formation and its possible interactions with GSTs may yield a better understanding of the reasons why increased GST activity affords protection towards the cytotoxic effect of DDP in cultured cells. 4. Acknowledgements This investigation was supported in part by USPHS grant CA27967 awarded by The National Cancer Institute. S.A. also_wishes to thank Dr. Don Powell, Chairman, Department of Internal Medicine for providing research funds and Dr. Y. C. Awasthi, Professor, Department of Human Biological Chemistry and Genetics for many helpful discussions and advice. 5. References Minna. J.D.. Pass, H., Glatstein. E. and Ihde. D.C. (1989) Cancer of the lung. In: Cancer principles and practice of oncology, 3rd ed.. pp. 591-705. Editors: V.T. Devita. S. Hellman and S.A. Rosenberg. J.B. Lipincott Company. Philadelphia. Hansen. H.H. and Kristjansen, P.E.G. ( 1991) Chemotherapy of small cell lung cancer. Eur. J. Cancer. 27. 342-349.

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