effects of 6-keto-prostaglandin e1 on ascitic hepatoma-130 in vivo comparison with chemotherapeutic...

PROSTAGLANDINS

EFFECTS OF 6-KETO-PROSTAGLANDIN E l ON ASCITIC HEPATOMA-130 IN VIVO COMPARISON WIT~ CHEMOTHERAPEUTIC AGENTS

Hiroshl Kobayashl *I , Masanori OJima*, Hiroshi Satoh**,

*Central Institute for Electron Microscopic Research. Nippon Medical School.

Sendagl, Bunkyo-ku, Tokyo, Japan.

**Sasakl Cancer Institute, Tokyo, Japan.

A b s t r a c t

The inhibitory effect of 6-keto-prostaglandin E l (pGEI) on the growth and survival of ascltlc hepatoma (AH-130) cells in vivo was compared with currently used chemotherapeutic agents. Three days after receiving an intraperltoneal injection of ~I06 AH-130 tumor cells, Donrhyu rats were injected intravenously or Intraperitoneally with one of the following: Thromboxane B 2 (TXB2) (0.5 mg/kg), 6-keto-pGE 1 (0.5 mg/kg), Mitomycln C (MMC) (1.5 mg/kg), or MMC + 6-keto-pGE I (1.5 mg/kg + 0.5 mg/kg).

The mean survival time, median survival time, and increase of llfe survival percent (ILS%) during a 60 day period revealed that both 6-keto-pGE I and 6-keto-pGE 1 + MMC significantly luhiblted AH-130 tumor cell growth, while TKB 2 promoted tumor cell growth.

We conclude that 6-keto-pGE 1 llke anti-tumor agents such as MMC, Diketocorlolln B, Carbazilquinon, Endoxan, and 5-Fluorouracil, can significantly inhibit growth of AH-130 tumor cells i_~nvivq, particu- larly when administered in combination with the anti-tumor agent MMC.

I n t r oduc t i on

It is well established that prostaglandins, formed by the metabolism of arachidonlc acid, exhibit potent anti-platelet aggregation

in vivo 1),2),3) It activity and can inhibit the metastatic process is not surprising, therefore, that 6-keto-PGFla which Is converted to 6-keto-pGE 1 via the 9-hydroxy prostaglandln dehydrogenase pathway in the liver and platelets, also exhibits a powerful anti-platelet aggregation effect. 4) However, the effect of this analogue, if any, on tumor growth and metastasis, has not been established. In this study, we examined the effect of 6-keto-pGE 1 on tumor cell growth l_~n vivo using AH-130 ascltes cells and compare these results with the

IAddress for Reprints: H. Kobayashl, M.D., Ph.D., Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 8ethesda Avenue, Cincinnati, Ohio 45267-0575, U.S.A.

FEBRUARY 1985 VOL. 29 NO. 2 255

PROSTAGLANDINS

conventional chemotherapeutic agents: Mitomycin C (MMC) and Diketocoriolln B (DKB), Carbazllqulnon, Endoxan, 5-Fluorouracll, pGD2, and pGl 2.

M a t e r i a l s and Methods

I. Animal: Female Donrhyu (DR) rats I0 weeks of age and weighing 160 g, were used as the experimental model (Charles River Company). Animals were housed in a specific pathogen free room with laml- nar flow racks.

2. Tumor Cells: AH-130 tumor cells were kindly supplied by the Sasaki Cancer Institute. Approximately 3x106 tumor cells were transplanted into the abdominal cavity by intraperltoneal injection in 3 ml. The time of injection is referred to as Day "0" throughout this study.

3. Thromboxane B 2 and 6-keto-prostaglaudin E l were kindly donated by the Ono Pharmaceutical Company and Mitomycln C was purchased from the Kyowa Hakko Company in Japan. The experimental schemes were as follows: TXB 2 (0.5 mg/kg), 6-keto-pGE 1 (0.5 mg/kg), diluted with 0.9% saline were injected intravenously in the tall vein and MMC ([.5 mg/kg) diluted with 0.9% saline was injected intraperitoneally beginning 3 days after the recipient animal received 3x106 AH-130 tumor ee!ls. The animals were divided into five groups as follows: TXB 2 was given daily for 8 days (group II), 6-keto-pGE 1 was give, daily for I0 days (group III), MMC was given once (group IV), and a single intraperitoneal injection of MMC on day 3 followed by intravenous injections of 6-keto-pGE 1 for I0 days (group V) and control animals which received intravenous injections with 0.9% saline injection (0.9 m!/kg) for I0 days (group I).

4. Evaluation of the Anti-tumor Effect: Three mlllileters of ascitic fluid was taken from each group of animals twice a week and immediately centrifuged at 800 rpm. The cells (pellet) were then fixed in 2.5% glutaraldehyde in phosphate buffer, pH 7.4 for 24-48 hours, followed by post fixa- tion in I% osmium tetroxlde for one hour. After dehydration with graded series of alcohol, the tissues were embedded in EPOCK 812.

The specimen block was sectioned with LKB ultramicrotome and stained with uranylacetate and lead citrate. The ultrastructural study was performed using the Hitachi-800 electron microscope. The increase of life survival percent (ILS%), median survival time, and mean survival time were determined up to 60 days following inoculation with AH-130 tumor cells.

256 FEBRUARY 1985 VOL. 29 NO. 2

PROSTAGLANDINS

5. Statistical Analysis Comparisons between groups were made according to the Mann-Whitney-Wilcoxson test (two sample rank test). Rejection of the null hypothesis was satisfied when p<.05. All data are expressed as the mean ± SD.

Results

I. The anti-tumor effect on AH-130 cells of the agents tested is shown in Table I. Increase median survival times were seen in animals treated with 6-keto-pGE 1 and MMC either alone or in combination while the median survival of animals receiving TXB 2 was decreased compared to untreated controls.

Test 100)%] , determined at the median The ILS% = [(Control × I00 -

survival time was: Group 1 0%; Group II -14.3%; Group III +23.0; Group IV +169.8; and Group V +205.6. Group V treated with MMC+6-keto-pGE 1 showed marked prolongation of survival rate based on this formula.

TABLE 1

Effects of 6-keto-pGEl, MMC and MMC + 6-keto-pGE 1 on rats inoculated with AH-130 tumor cells.

Durations of Median Mean In vivo treatment survival survival

Grou~ # n treatment in vivo days ILS % days*

I 6 Control I0 12.6 0 13.17±0.98 II 6 TKB 2 8 10.8 -14.3 II.50~1.51

III 5 6-keto-pGE 1 I0 15.5 +23.0 15.60~3.84 IV 6 MMC I 34 +169.8 44.50t17.43 V 5 MMC + 1 38.5 +205.6 41.40~18.71

6-keto-pGE 1 I0

*Means ± SD n = number of animals per experiment.

2. As shown in Table 2, untreated rats receiving AH-130 tumor cells intraperitoneally (Group I) survived 13.17±0.98 days and TXE 2 treatment (Group II) slightly shortened survival time to 11.50±1.51 days (P<.08 vs. Group I). Group III receiving 6-keto-pGE 1 survived 15.60~3.84 days, which is not statistically different from Group I. Treatment with MMC (Group IV) alone or in combination with 6-keto-pGE 1 (Group V) significantly pro- longed mean survivals to 44.50~17.43 days (P<.O06) and 41.40±18.71 days, P<.01), respectively.


PROSTAGLANDINS

TABLE 2

Effect of TXB2, 6_keto_pGEl ' MMC ~nd MMC + 6-keto-pGE I on survival of Donrhyu rats with AH-130 tumor cells.

6-keto-pGE 1 +

Animal # Control* TKB2* 6-keto-pGEl* MMC* MMC*

1 12 9 I0 22 17 2 13 II 14 31 31 3 13 II 17 34 39 4 13 12 18 60 60 5 13 13 20 60 60 6 15 13 60

I 6) 1

*Number of days. Brackets indicate two groups compared: I) p<.08 2) p<.86 3) p<.18 4) p<.04 5) p<.006 6) p<.0t

3. The ultrastructural findings in the ascitic fluid in each group are shown in Fig. I. The normal AH-130 cell contains a pseudonuclear inclusion body which accompanies the tubular structure

n 5) The nuclear membrane inva- surrounding the nuclear membra e. ginates into the nucleoplasm and became continuous with the rough endoplasmic retlculum (arrow) (Plate #I). Cells from animals treated with TXB 2 had an increase in the number of ribo- somes, mitochondria, and rough-end-plasmic reticulum (Plate #2). In addition, marked increases in the number of mlcrovilll and mlcrofilaments, development of the Golgl-complex and dilation of the rough endoplasmlc retlculum were noted (arrows).

Hepatoma cells from 6-keto-pGE 1 treated animals (Plate #3), showed a disappearance of the mlcrovilll, marked degeneration of the nuclear inclusion body, swelling of the outer membrane of the mitochondrla, degeneration of the mitochondrial crlstae, and an increase of lyosomes (arrow).

Cells from MMC treated animals (Plate #4), showed aggregation of chromatln in the nuclear membrane, an increase of hetero- chromatln, and swelling of mitochondrla. In addition to the changes seen following MMC above, in animals treated wlth MMC + 6-keto-pGE 1 (Plate #5), the tumor cells became aggregated, the nuclear chromatin was clumped, and lysosomal complexes ware increased.


PROSTAGLANDINS

Figure I.

Plate #I: The invagination of the inner nuclear membrane into the nucleoplasm (arrow) is noted in AH-130 tumor cells as well as the presence of numerous micro-villi in Group I. x9000.


PROSTAGLANDINS

Figure I.

Plate #2: A dramatic increase of microfilaments, marked development of the Golgl-complex (arrows), and dilated rough endoplasmic retlculum are noted in cells from Group II. x6000.

i


PROSTAGLANDINS

F_i~ure 1.

Plate #3: The marked increase of secondary lysosome, lipid, and the degeneration of the pseudo-nuclear inclusion bodies are noted and disappearance of micro-villl (arrow) is typical of cells in Group III. xg000.


PROSTAGLANDINS

Figure I.

Plate #4: The marked aggregation of chromatin in the nuclear membrane, and swelling of the mltochondria are noted in Group IV. ×9000.


PROSTAGLANDINS

Figure .I •

Plate #5: The marked increase of the lysosomes, clumping of the chromatln in the nucleoplasm are noted in Group V. x6000.


PROSTAGLANDINS

Discuss ion

The ultrastructural study disclosed that the AB-130 tumor cell projects microvllli from its surface and contains a pseudonuclear inclusion body in the nucleoplasm surrounded by the nuclear membrane. Ribosome granules, mltochondra, rough endoplamic reticulum, and Golgi-complexs are clearly seen in the cytoplasm. When 6-keto-pGE 1 was administered (Group II), mlcrovilli disappeared, ribosome granules degenerated, the number of lyaosomes increased, and both mlto- chondrla and nuclear inclusion bodies underwent degeneration. The AN-130 tumor cells treated with 6-keto-pGE 1 lost their glyocalyx layer and became swollen. The possible mechanisms by which 6-keto-pGE 1 exerts this anti-tumor effect may include inhibition of the Na+-K+-ATPase and/or Ca++-Mg++-ATPase enzymes located in the subplasma membrane leading to altered membrane permeability and even- tual cell swelling. The increase in the number of lysosomes in tumor cells subsequent to 6-keto-pGE 1 administration suggests that they may be important to the anti-tumor action of prostaglandin. However, we should llke to emphasize that conclusive evidence of the precise mechanisms is not currently available.

Other prostaglandins such as pGD 2 have been shown to possess antl-tumor action and this effect may be linked to their platelet anti-aggregatory action. The antl-aggregatlon action is associated with an increase in cellular cyclic AMP. 2) Strlngfellow 14) demon- strated that cells which produce low pGD 2 levels have a higher metastatic potential than do cells with high pGD 2. He concluded that exogeneoualy added pGD 2 inhibited tumor metastasis due to an anti- aggregatory action on the tumor cell. In addition to these actions, Simmet and Jaffe 15) recently reported a probable cytotoxlc effect when they showed that pGD 2 inhibited tumor growth in a dose dependent manner using a B-16 melanoma cell llne in vitro. DNA, RNA, and pro- tein synthesis were all inhibited at high pGD 2 doses. However, Fukushlma 13) reported that 9-deoxy-Ag-pGD 2 (pGJ 2) derived by removal of one singlet oxygen from pGD 2 had less anti-aggregatory action than pGD 2 but retarded growth of cultured LI210 leukemia cells 3 times more than did pGD 2.

Thromboxanes, are the terminal products in the prostaglandin pathway and one of them, TXB2, has been documented to enhance cell growth. Honn 16) reported that 3H-thymldine uptake of B-16 melanoma cells increased after adding TXB 2. This was associated with a lower cyclic AMP level. He also reported that U46619 (UpJohn Pharmaceu- tical Co.), a stable analogue of TKA2, promotes cell growth. The marked development of the Golgi-complex, augmentation of mlcroEila- menta, and dilatation of rough endoplasmic retlculm noted in our study (Group II. treated with TXB 2) are consistent with and add ultraatructural documentation to these observations of Honn.


PROSTAGLANDINS

The exact role of cyclic AMP in tumor growth and development is unclear, however, it has been shown that ATPase activity of the tumor cell membrane is higher than that of normal cell membrane 6) and the cyclic AMP level of the tumor cell membrane is lower than that found in the normal cell. 7), 8), 9) In addition, it has been proposed that Diketocoriolln B, I0) and anti-body to tumor cell ATPaae, exert their anti-tumor activity by inhibiting tumor cell ATPase act[vlty. Alkylatlng antl-tumor agents can inhibit ATPase activity and increase the cyclic AMP level. Tumor cells which demonstrate a pleiotroplc drug resistance to antl-tumor alkylating agents are reported to have an increased ATPase actlvlty. II)

Assuming that MCC, 12) Carbazilquinon, Endoxan, 5-Fluorouracil,

Diketocoriolin B,pGI2, pGD2, and 6-keto-pGE 1 exert their anti-tumor effect through a common mechanism, we examined the structure of these agents in an attempt to find a common denominator. We found that this group of compounds based on a similarity of the ~ atom charge separation would fit a putative receptor with three binding points. The receptor model and the planar structures of these agents (Table 3) require that these three points, one positively charged and two negatively, be separated as follows: 5.0~-0.3 A between the two negatively charged sites, 3.7±0.2 A between one negative site and the positively charged site, and 6.1±0.3 A between the second negative site and the positive site. We also determined that 6-keto-pGE 1 had a chemical structure and charge distribution that could accommodate the proposed receptor model.

Certainly the removal of oxygen from the cyclopentane ring of pG is enough to induce an anti-tumor action. It has been reported that 9-deoxy-A9-pGD2, produced by removing one oxygen from the cyclopentane ring of pGD2, has a high anti-tumor activity. It is interesting as well that removal of the ring oxygen from pGD 2 results in a con- siderable reduction in the occurance of dlarrhea. 13) According to this scheme, it might be reasonable to expect that 9-deoxy-6-keto- pGEI, produced when oxygen is removed at the 9th position from 6-keto-pGEl, would possess both anti-tumor and platelet anti- aggregatory actions and as seen with PGD2, the removal of this oxygen from 6-keto-pGE 1 reduces the occurrence of diarrhea and anti- hypertensive action.

These results provide new suggestive evidence that the anti- tumor actions of 6-keto-pGE 1 are shared with the more traditional anti-tumor agents. The precise reason for this similarity may reside in common site and mechanlsm(s) of action. Animals treated with 6-keto-pGE 1 survived nearly 25% longer than untreated animals. The addition of 6-keto-pGE 1 to MMC improved survival over MMC treatment alone. It therefore appears that 6-keto-pGEl, an extremely stable i__nn vlvo prostaglandin, may be an important endogenous tumor cell suppressent.


P R O S T A G L A N D I N S

T&BLE 3

Structural activity relationship between prostaglandlns and anti- tumor agents,

H=C OCH, O -

. " ~ - ~ " , o . 'OH +2 0 0 +

Mitomycin C 6-keto-PGE i

I I I I t'"~--~, "'~"'-~,,-"' ~ o. .~ . . . . _o +

P G 12

Carbazilquinon o OH

0 C H ~'cHt "el

J ~'C H ~ -CH p-Ci OHz~ o-

+ PG D~

Endoxan + *

OH+ F (C OH)

'O O

o ~ L ~ / C ~ C H ~ (C. OH. NH) 6 I±0 3~ (=O Cl F)

~'o-~c. , , .o~ , + + , . . . . o . . . . ,o~ ; - P' p

Oike tocor io l in B

2 6 6 F E B R U A R Y 1 9 8 5 V O L . 2 9 N O . 2

PROSTAGLANDINS

Acknowledgments

We thank Ono-pharmaceutical Company for providing us with TXB2, 6-keto-pGE I. We are also grateful to Dr. Ronald W. Millard, Head of Cardiovascular Pharmacology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, U.S.A. for help in preparing the manuscript. This work was supported in part by Adult Disease Clinic Memorial Foundation, Shlbuya, Tokyo, in 1983.

References

I. Honn, K.V., B. Cicine, and A. Skoff. Prostacyclin: A Potent Antimetastatic Agent. Science 212:1270. 1981.

2.

3.

Honn, K.V., W.D. Busse, and B.F. Sloane. Prostacyclin and Thromboxanes. Implications for their Role in Tumor Cell Metastasis. Biochem. Pharmacol. 32:1. 1983.

Honn, K.V., J. Meyer, G. Neagos, T. Henderson, C. Wesdey, and V. Ratanatharathorn. Control of Tumor Growth and Metastasis with Prostacyclin and Thromboxane Synthetase Inhlbltors: Evidence for a New Anti-tumor and Antl-metastatic Agent (Bay g 6575). In: Interaction of Platelets and Tumor Cells. (G.A. Jamison, ed.) Alan R. Liss, New York, 1982. p. 295.

4.

5.

6.

7.

Wong, P.Y-K., W.H. Lee, P.H-W. Chao, R.F. Russ, and J.C. McGiff. Metabolism of Prostacycli~ by 9-hydroxy Prostaglandin Dehydro- genase in Human Platelets. J. Biol. Chem. 255:9021. 1980.

= .

Hoshino, M. The Deep Invaglnation of the Inner Nuclear Membrane into the Nucleoplasm in the Ascites Hepatoma Cells. Exp. Cell. Res. 24:606. 1961.

Kasarov, L.B., and H. Friedman. Enhanced Na-K-actlvated Adenosine Triphosphatase Activity in Transformed Fibroblasts. Cancer Res. 34:1862. 1974.

Hickle, R.A., C.M. Walker, and A. Datta. Increased Activity of Low-Km Cyclic Adenosine 3-5-monophosphate Phosphodiesteraae in Plasma Membranes of Morris Hepatoma 5123 tc(h). Cancer Res. 35: 601. 1975.

8.

9.

Hickie, R.A., S.H. Jan, and A. Datta. Comparative Adenylate Cyclase Activities in Homogenate and Plasma Membrane Fractions of Morris Hepatoma 5123 tc(h). Cancer Res. 35:596. 1975.

Tisdale, M.J., and B.J. Phillips. Inhibition of Cyclic 3,5-nucleotidephosphodiesterase--A Possible Mechanism of Action of Bifunctlonal Alkylat[ng Agents. Biochem. Pharmacol. 24:211. 1975.


PROSTAGLANDINS

I0.

II.

12.

13.

14.

Kunlmoto, T., M. Hori, and N. Umezawa. Mechanism of Action of Diketocorlolin B. Biochlm. Biophys. Acts 298:513. 1973.

Tsukagoshi, S., A. Moriwakl, and Y. Sakurai. Increase of Adenosine Triphosphatase Activity of Yoshlda Sarcoma Cells in the Process of Acquiring Resistance o~ Alkylating Agents. Gann. 62:65. 1971.

Sybalski, W., and V.N. lyer. Crosslinklng of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics. Proc. 23:946. 1964.

Fed.

Fukushima, M., T. Kato, K. Ota, Y. Arai, S. Narumlya, and O. Hayaishi. 9-Deoxy-prostaglandin D2, a Prostaglandin D 2 Derivative with Potent Antlneoplastlc and Weak Smooth Muscle- contracting Activities. Biochem. Biophys. Res. Commun. 109:626. 1982.

Stringfellow, D.A., and F.A. Fitzpatrick. Prostaglandin D 2 Controls Pulmonary Metastasis of Malignant Melanoma Cells. Nature 282:76. 1979.

15. Simmer, T., and B. Jaffe. Inhibition of B-16 Melanoma Growth in Vitro by Prostaglandin D 2. Prostaglandins 25:47. 1983.

16. Honn, K.V., and J. Meyer. Thromboxanes and Prostacyclin: Positive and Negative Modulators of Tumor Growth. Biochem. Biophys. Res. Commun. 102:1122. 1981.

Editor: Peter W. Ramwell Received: 4-10-84 Accepted: 11-25-84


effects of 6-keto-prostaglandin e1 on ascitic hepatoma-130 in vivo comparison with chemotherapeutic...

Documents