Inhibition of motility and invasion of human lung cancer cells by invasion inhibiting factor 2

Surgical Oncology 1996; 5: 77-84

Inhibition of motility and invasion of human lung cancer cells by invasion inhibiting factor 2 Z. G. HAN,* W. G. JIANG,t S. HISCOX,t M. B. HALLETT,t A.ISOAlt AND R. E. MANSELt *Department of Thoracic Surgery, Third Teaching Hospital, Norman Bethune University of Medical Sciences, Changchun Jilin 130031, China; tUniversity Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK; and t-Research Centre, Asahi Glass Co. Ltd, 1150 Hazawa-cho, Kanagawa-ku, Yokohama 221, Kanagawa, Japan

The motility and invasion of cancer cells are basic requirements for the establishment of distant metastases. In this study, we examined the effect of invasion inhibiting factor 2 (IIF2), a motility/invasion regulatory agent, on the motility, invasion, growth and basement membrane attachment of human lung cancer cells. IIF2 significantly reduced cell dissociation, colony scattering and invasion induced by the motogenic factor, HGF/SF. Western and Northern analyses showed these cells to be positive for the HGF/SF receptor comet. These effects were blocked by an anti-IIF2 antibody. IIF2 did not affect the growth and attachment of lung cancer cells to the basement membrane. It is concluded therefore that invasion inhibiting factor 2 is an inhibitor of human lung cancer cell motility and invasion in vitro and this may bear some importance in the construction of anti-metastatic therapies. Surgical Oncology 1996; 5: 77-84.

Keywords: cell invasion, cell motility, hepatocyte growth factor/scatter factor (HGF/SF), invasion inhibiting factor 2 (IIF2), lung cancer cell.

including tumour invasion inhibiting factor 2 (IIF2), have been reported to have anti-motility and antiinvasion effects on malignant cells [7-12]. IIF2 was originally identified in bovine liver and has been shown to possess an anti-invasive property in vitro. IIF2 has also been reported to prevent lung metastasis from melanoma and Lewis lung carcinoma cells in animal models [10-13]. Lung cancer is one of the commonest tumours in the world. The presence of metastatic deposits in patients suffering from lung cancer greatly decreases their prognosis. Many patients possess multiple metastatic foci undetectable at the clinical diagnosis of primary tumour-surgical resection for local lesions is therefore of limited value [14-17]. It is therefore important to search for strategies aimed at reducing the metastatic potential of these cancer cells. In this study, we have examined the in vitro effects of IIF2 on human lung cancer cell growth, basement membrane attachment and HGF/SF-induced cell motility and invasion.

INTRODUCTION Tumour cell metastasis is the major factor affecting the prognosis of cancer patients. In order for cancer cells to establish distant metastases, they must detach from the primary tumour, degrade and migrate through the basement membrane and extracellular matrix, intravasate, and travel in the circulation to the new site where reattachment and extravasation occur, leading to the development of a new focus [1-4]. Cancer cell motility and invasiveness are events that can be triggered by a variety of tumour motility factors including hepatocyte growth factor/scatter factor (HGF/SF) [5, 6]. The motility and invasive properties of cancer cells have therefore become targets for anti-metastatic treatment. In the past few years some agents, Correspondence: Dr W. G. Jiang, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. © 1996 Blackwell Science Ltd

77

78

Z. G. Han et al.

MATERIALS AND METHODS Cell lines and cytokines The human lung cancer cell lines SK-MES 1 (squamous cell carcinoma) and COR-L23 (large cell carcinoma) were obtained from the ECACC (European Collection of Animal Cell Culture, Salisbury, UK) and maintained in DMEM and RPMI 1640 with 10% FCS (Sigma, UK) respectively. Matrigel (reconstructed basement membrane) was purchased from Collaborative Research Products (Bedford, MA). Cytodex-2 carrier beads were from Pharmacia. Invasion inhibiting factor 2 was synthesized as described previously [11], and a rabbit anti-IIF2 polycolonal antibody was raised as reported [12] and was found to be specific to the IIF-2 used in this study [12, 13, 18]. Human recombinant hepatocyte growth factor (HGF) was a gift from Dr T. Nakamura, Osaka, Japan. A mouse antihuman HGF receptor (the product of comet oncogene) antibody for Western blotting was purchased from TCS (UK). All other materials were purchased from Sigma, UK, except where mentioned.

Colony scattering Lung cancer cells were seeded into 96-well plates at 4 x 103 per well and left overnight to allow colony formation [19]. IIF2, HGF, or a combination of the two were then added onto the cells which were incubated for a further 24 h. The cells were then fixed and stained with crystal violet prior to being photographed.

Cell dissociation assay Cells were cultured with cytodex-2 carrier beads (Pharmacia, prepared at 25 mg mr ' in BSS as stock and used at 5 mg mr') for 24 h and then harvested and washed in culture medium [20]. The cell number was adjusted to 5 x 105 per ml- 1 and the suspensions of beads, together with attached cells, were placed into 96-well plates (Nunc, Denmark). The testing reagents were then added to the cells and incubated for 24 h. Following this, the plates were then emptied and washed with BSS buffer to remove all the carrier beads and unbound cells. The cells which had detached from the beads

and bound to the bottom of the wells were fixed in buffered formalin and stained with 0.5% crystal violet. These stained cells were then counted with a light microscope (four fields per well). Cell growth Cells were seeded into 96-well plates at 105 mr ' and IIF2 was added. Plates were cultured for 72 h and cell numbers were measured using the MIT assay [20]. The crystals produced were extracted with 10% Triton X-100 and the absorbance measured at 540 nm. The cell growth was shown as a percentage of growth induced by IIF2 compared with control (culture medium only). Cell attachment assay This was based on a recently described method [21]. Briefly, Matrigel (1 I1g per well) was added to multi-well plates, which were incubated for 24 h to allow binding to the surface of the well. The plates were then washed, and bovine serum albumin (BSA) (5% w/v) was added to block the remaining binding sites. Cells were seeded at 104 per well with or without IIF2 for 30 min and unbound cells removed by aspiration. The numbers of attached cells were measured by the 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MIT) assay. Basement membrane invasion assay This was based on a method reported by Albini et al. and Parish et al. [22, 23]. 24-well culture plates were equipped with tissue culture inserts (Falcon Dickinson, USA). One end of the insert was sealed by a polycarbonate filter of pore size 8.0 11m to allow cells to migrate. Matrigel (50 I1g per membrane) was then added to each filter and incubated for 24 h at 3rC for gel layer formation. The Matrigel layer was rehydrated and the cells were the added into each insert at 5 x 104 per insert. To these wells were then added 1 ml of medium, either with or without HGF/SF (50 ng per mr'), IIF2 (10 I1g mr ') and anti-IIF2 antibody (10 I1g mr') and incubated at 3rC for 24 h. The cells on the upper surface of the filter were then completely removed by wiping with a cotton swab; the cells which had migrated through the Matrigel and attached to the underside of the filter were then fixed, stained and counted. ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84

IIF2 and lung cancer invasion

79

3.5

Western blotting of cell protein 3.0

Cell proteins were extracted as previously described [25]. The samples were then subjected to SOS-PAGE and Western-blotted onto a nitrocellulose membrane. Nonspecific binding sites were blocked by 10% skimmed milk protein for 1 h. Following incubation with primary antibody (mouse monoclonal anti-comet) and secondary antibody (anti-mouse IgG horseradish peroxidase conjugate), the blots were developed using an enhanced chemiluminescence system (ECl, Amersham, UK).

)(

" ::>-

SK-MES 1 .=+HGF A=Control

2.5

"l:I

.5

2.0

'0:;

1.5

0

~

1.0 0.5 0 0

0.1

IIF2 Jlg

Northern blot hybridization Total cellular RNA was prepared from cultured cells using a method of extraction based on that described by Chomczynski & Sacchi [26]. For the Northern blot analysis, 10 pg of RNA from each cell sample was fractionated by electrophoresis on 0.8% denaturing agarose gels and transferred to a nylon membrane. Hybridization using an entire comet cONA clone was carried out at 45°C in the presence of 50% formamide. Blots were washed twice in 2 x SSC-O.l % SOS at room temperature for 15 min, and once at 45°C for 15 min. Errors in sample loading were corrected by probing each membrane for human f3-actin.

10

100

mr'

Figure 1. Concentration-dependent inhibition of cell motility by Cytodex-2 dissociation assay. The data shown is for the dissociation of SK·MES 1 cells, treated with IIF2 in the presence or absence of HGF/SF (10 ng rnl').

3.0

)(

2.5

"

"l:I

.5

>- 2.0 ::E .+: 0

~

1.5

1.0

RESULTS

Control

HGF

HGF+IIF2 HGF+IIF2+abIlF2

Effect of IIF2 on cell motility IIF2 inhibited the HGF-induced dissociation of the lung cancer cell lines SK-MES 1 and COR-l23 from carrier beads in a concentration-dependent manner over the range 0.5-50 pg mr ' (Fig. 1). The effects of IIF2 were blocked by anti-IIF2 antibody (Fig. 2). IIF2 also inhibited the HGF induced scattering of colonies of COR-l23 cells (Fig. 3).

3.0

)(

2.5

CD

"l:I

.5 ~

2.0

'+:

o

~

1.5

Regulation of cell growth by IIF2 1.0

In order to determine whether the observed reduction in motility after IIF2 treatment could be attributed to changes in cell growth rate, cell growth was measured. IIF2 (up to 40 pg mr') did not affect growth over a 3-day culture period (data not shown). This observation was similar to those reported previously for other cell lines [12, 13]. ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84

Figure 2. The effect of anti-IIF2 antibody on the IIF2 inhibition of cell motility. HGF/SF (40 ng ml-')-induced motility was measured after treatment with IIF2 (10 IIg rnl"] or IIF2 (10 Jlg ml :") plus anti-IIF2 antibody (40 ng rnl'). The data shown are for COR-l23 and SK-MES 1 cells.

80

Z. G. Han et al.

(a)

(b)

(d) Figure 3. IIF2 inhibition of HGF/SF-induced colony scattering of COR-L23 cells. (a) Colonies of COR-L23 cells in culture; (b) after treatment with HGF/SF (50 ng/ml); (c) in the presence of IIF2 (10 Jig ml-') and HGF/SF; (d) cells with IIF2 (10 JIg

rnl" ') alone.

Thus, IIF2 inhibits cell motility without affecting cell growth.

Analysis of lung cancer cell HGF/SF receptor status

Effect of IIF2 on cell attachment

Western blotting studies of lung cancer cell proteins revealed the presence of a 190 kDa mature HGF/SF receptor protein, together with the 145 kDa

Culture of the two lung cancer cell lines with a

comet p-L1nit in the cell lines (Fig. 6). These results

range of concentrations of IIF2 (0.3-40 Jig mr': had no effect on their attachment to the artificial basement membrane, Matrigel, compared to the control cells (incubated in medium alone) (Fig. 4).

Basement membrane invasion assay

were further confirmed by Northern analysis of cellular mRNA, which demonstrated the presence of an 8 kb comet transcript in the two cell lines (Fig. 7).

DISCUSSION

Inclusion of IIF2 in the invasion assay significantly

The motility and invasiveness of cancer cells are

decreased HGF/SF-induced SK-MES 1 invasion into the artificial basement membrane, Matrigel,

key elements in the establishment of metastatic lesions, the most important factor in determining

compared with their controls. Inclusion of the antiIIF2 antibody significantly blocked these effects (Fig. 5).

the survival of patients with cancer. Many cytokines have been reported to play important roles in tumour progression and in the development of ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84

IIF2 and lung cancer invasion 160

9.0 7.5

128 Ql

C

Ql

-a

COR-L23

96

u

III III

-i

I-

64

• .--.

>- 4.5

..

~ 0

::E

~

32

0

"

0.0 0.Q1

10

1

0.1

IIF2 f!Q ml-

100

128

SK-MES 1

c

96

----..

.&:

III OJ OJ

IV

•

~

64

IIF2

•

HGF +IIF2

HGF +IIF2 +ab-IIF2

Figure 5. Basementmembrane invasion assay. The number of cells invading through an artificial basement membrane in response to HGF/SF was determined and is shown here as an invasion index. IIF2 was seen to significantly decrease the invasive ability of SK-MES 1 cells. Inclusion of an anti-IIF2 antibody blocked these effects. HGF/SF was used at 50 ng ml-', IIF-2 10 /1g mr', and anti-IIF-2 antibody 10 /1g ml- 1 •

OJ

u

control HGF

1

160

Ql

3.0 1.5

0

E

6.0

.5

.&:

..

SK-MES1

><

OJ

E

81

~

32

0 0

0.01

0.1

1

IIF2 f!Q ml-

10

100

1

Figure 4. The effects of IIF2 on cell attachment to the artificial basement membrane, Matrigel. as determined by MTI assay. IIF2 had no significant effect on cell-matrix attachment.

metastases [26-30]. HGF/SF is one such protein

en I o~

and has been reported to increase the motility and invasiveness of various carcinoma cells [6, 31]. The receptor for HGF/SF has been reported to be a 190 kDa heterodimer, consisting of a 50 kDa-a and a 145 kDa-p subunit coded by the c-met protooncogene [32]. A number of normal and malignant cell types are known to express this receptor protein [23, 33]. In the past few decades, great efforts have been made to understand mechanisms of the metastatic spread of tumours and to search for suitable strategies that can reduce the formation of these lesions [35-37]. It has been demonstrated that inhibitors of tumour cell invasion in vitro are useful in the prevention of cancer metastasis in vivo. Invasion ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84

Figure 6. c-met receptor expression was detected by Western blotting. The two lung cancer cell lines expressed an approximate 190 kDa and/or 145 kDa protein as identified by a specific anti-human c-met f3 subunit antibody.

82

Z. G. Han et al.

I

ex

Figure 7. Northern blot analysis of c-met message. 10 J1g

of cellular RNA was blotted onto a nylon membrane and hybridized with a full length cDNA for c-met. The cell lines showed presence of an 8 kb transcript corresponding to the common c-met message. Errors in sample loading were corrected by re-probing each membrane for human f3-actin.

inhibiting factors have been shown to have antimetastatic properties in animal models of melanoma and lung metastasis [10-13]. In addition, IIF2 has also been reported to inhibit the motility and invasion of human colon cancer cells [18]. In this study, we report that IIF2 inhibited HGF/SF-induced colony scattering, dissociation and invasion through the basement membrane of human lung cancer cells. IIF2 did not inhibit the cell growth or attachment of human lung cancer cells to the extracellular matrix. These results, along with others [10-12] suggest that IIF2 does not inhibit the binding of tumour cells to matrix components. The cellular and molecular mechanism for the antimotility and anti-invasion properties of IIF-2 is not yet established. We have previously shown that this inhibition is independent of changes in E-cadherin, fibronectin receptor and cytosolic free

calcium; vimentin and actin are also unchanged in IIF-2 treated cells [18]. Clearly, more work in this area is now required to explore other pathways, for example, IIF2-cMET interaction, protein kinase C and other signalling pathways. HGF/SF has been implicated in the process of cancer cell metastasis due to its stimulatory effects on the metastatic functions of tumour cells. Kaneko et al. [38] has shown that patients with liver metastasis had a higher level of circulating HGF/SF than normal controls and that these levels were raised further after the resection of metastatic foci. Pleural exudate from patients with lung and pleural metastases has also shown high HGF/SF levels [39]. Even routine surgical operations may induce significant increases in blood HGF/SF levels [40]. Taniguchi et al. [41] has reported that the increase in blood HGF/SF levels of breast cancer patients is signifcantly associated with axillary lymph node metastasis. It is therefore possible that by lowering the reactivity of cancer cells to HGF/SF, their metastatic potential may be suppressed. Various agents such as IL-12 and y-linolenic acid have been shown to inhibit HGF/SF-induced motility and invasion [42,43]. We have shown here that IIF2 reduces HGF/SF-induced motility and invasion of human lung cancer cells. These results suggest that IIF2 might be therapeutically useful to prevent accidental seeding of tumour cells during the surgical removal of primary tumours. Further investigation of the in vivo effects of IIF2 will be of use in determining its suitability as a potential antimetastatic agent.

ACKNOWLEDGEMENTS The authors would like to acknowledge support from the British Council, the Welsh Scheme for the Development of Social and Health Research (WSDSHR), and Scotia Pharmaceuticals Ltd (UK).

REFERENCES 1. Liotta LA. Biochemical mechanisms of tumour invasion and metastases. Clin Physiol Biochem 1987; 5: 190-9. 2. Miller FR. Immune mechanisms in the sequential steps of metastasis. Crit Rev Oncogenesis 1993; 4: 293-311. ©1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84

IIF2 and lung cancer invasion 3. Liotta LA, Stracke ML. Tumour invasion and metastasis: biochemical mechanisms. Cancer Treat Res 1988; 40: 223-38. 4. Schiffmann E. Motility as a principle reqirement or metastasis. Cancer Invest 1990; 8: 673-4. 5. Stoker M, Gherardi E, Perryman M, Gray J. Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 1987; 327: 239-42. 6. Jiang WG, l.lovds D, Puntis MCA, Nakamura T, Hallett MB. Regulation of spreading and growth of colon cancer cells by hepatocyte growth factor. Clin Exp Metastasis 1993; 11: 235-42. 7. Melchiori A, Albini A, Ray JM, Stetler-Stevenson WG. Inhibition of tumour cell invasion by a highly conserved peptide sequence from the matrix metalloproteinase enzyme prosegment. Cancer Res 1992; 52: 2353-6. 8. Mohler JL, Broskie EN, Ranparia DJ, Sharief Y, Coleman WB, Smith GJ. Cancer cell motilityinhibitory protein in the Dunning adenocarcinoma model. Cancer Res 1992; 52: 2349-52. 9. Ikeyama S, Koyama M, Yamaoko M, Sasada R, Miyake M. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J Exp Med 1993; 177: 1231-7. 10. Isoai A, Giga-hama Y, Shinkai K, Mukai M, Akedo H, Kumagai H. Tumour invasion-inhibiting factor 2: primary structure and inhibitory effect on invasion in vitro and pulmonary metastasis of tumour cells. Cancer Res 1992; 52: 1422-6. 11. Isoai A, Goto-Tsukamoto H, Murakami K, Akedo H, Kumagai H. A potent anti-metastatic activity of tumour invasion-inhibiting factor 2 and albumin conjugate. Biochem Biophys Res Commun 1993; 192: 7-14. 12. Isoai A, Goto-Tsukamoto H, Yamori T, et al. Inhibitory effects of Tumour Invasion-inhibiting Factor 2 and its conjugate on disseminating tumour cells. Cancer Res 1994; 54: 1264-70. 13. Isoai A, Giga-hama Y,Shinkai K, Mukai M, Akedo H, Kumagai H. Purification and caracterization of tumour invasion-inhibiting factors. Jpn J Cancer Res 1990; 81: 909-14. 14. Castonguay A. Methods and strategies in lung cancer control. Cancer Res 1992; 52: 2641s-51s. 15. Cancer research campaign. Lung cancer and smoking-UK. Factsheet, Cancer Research Campaign, 1992; 11. 16. Rubin SA. Lung cancer: past, present, and future. J Thorac Imaging 1991; 7: 1-8. 17. Kohn EC, Liotta LA. Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res 1995; 55: 1856-62. 18. Jiang WG, Hiscox S, Singhrao SK, et al. Inhibition of motility and invasion by invasion inhibiting factor 2 (IIF2) on human colon cancer cells. Surg Res Commun 1995; 17: 67-78. 19. Gherardi E, Gray J, Stoker M, Perryman M, Furlong R. Purification of scatter factor, a fibroblast-derived basic ©1996 BlackwellScience Ltd, Surgical Oncology, 5: 77-84

83

protein that modulates epithelial interactions and movement. Proc Natl Acad Sci USA 1989; 86: 5844-8. 20. Rosen EM, Meromsky L, Setter E, Vinter DW, Goldberg ID. Ouantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads. Exp Cell Res 1990; 186: 22-31. 21. Tada H, Shiho 0, Kuroshima K, Koyama M. Tsukamoto K. An improved colorimetric assay for interleukin-2. J Immunol Meth 1986; 93: 157-65. 22. Furukawa T, Watanabe M, Kubota T, et al. Significance of in vitro attachment of human colon cancer to extracellular matrix proteins in experimental and clinical liver metastasis. J Surg Onco/1993; 53: 10-6. 23. Albini A, Iwamoto Y, Kleinman HK, et al. A rapid in vitro assay for quantitating the invasive potential of tumour cells. Cancer Res 1987; 47: 3239-45. 24. Parish CR, Jakobsen KB, Coombe DR. A basement membrane permeability assay which correlates with the metastatic potential of tumour cells. Int J Cancer 1992; 52: 378-83. 25. Rygaard K, Nakamura T, Spang-Thomsen M. Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts. Br J Cancer 1993; 67: 37-46. 26. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 1987; 162: 156-9. 27. Yoshida K, Ozaki T, Ushijima K, Hayashi H. Studies on the mechanisms of invasion in cancer. l. Isolation and purification of a factor chemotactic for cancer cells. Int J Cancer 1970; 6: 123-32. 28. Liotta LA, Mandler R, Murano G, et el. Tumour cell autocrine motility factor. Proc Natl Acad Sci USA 1986; 83: 3302-6. 29. Atnip KD, Carter LM, Nicolson G L, Dabbous MK. Chemotactic response of rat mammary adenocarcinoma cell clones to tumour-derived cytokines. Biochem Biophys Res Commun 1987; 146: 996-1002. 30. Mizoguchi H, Komiyama S, Matsui K, et al. The response to epidermal growth factor of human maxillary tumour cells in terms of tumour growth, invasion and expression of proteinase inhibitors. Int J Cancer 1991; 49: 738-43. 31. Mooradian DL, McCarthy JB, Komanduri KV, Furcht LT. Effects of transforming growth factor-dl on human pulmonary adenocarcinoma cell adhesion, motility and invasion in vitro. J Natl Cancer Inst 1992; 84: 523-7. 32. Rosen EM, Knesel J, Goldberg ID, et al. Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumour. Int J Cancer 1994; 57: 706-14. 33. Bottaro DP, Rubin JS, Faletto DL, et al. Identification of the hepatocyte growth factor receptor as the c-Met

84

Z. G. Han et al.

proto-oncogene product. Science 1991; 251: 802-4. 34. Prat M, Narsimhan RP, Crepaldi T, Nicotra MR, Natali PG, Comoglio PM. The receptor encoded by the human comet oncogene is expressed in hepatocytes, epithelial cells and solid tumours. Int J Cancer 1991; 49: 323-8. 35. Crowley CW, Cohen RL, Lucas BK, Liu G, Shuman MA, Levinson AD. Prevention of metastasis by inhibition of the urokinase receptor. Proc Natl Acad Sci USA 1993; 90: 5021-5. 36. Collins JF, Herman P, Schuch C, Bagby GC Jr. C-myc antisense oligonucleotides inhibit the colony-forming capacity of colo 320 colonic carcinoma cells. J Clin Invest 1992; 89: 1523-7. 37. Schwartz GK, Jiang J, Kelsen D, Albino AP. Protein kinase C: a novel target for inhibiting gastric cancer cell invasion. J Natl Cancer Inst 1993; 85: 402-7. 38. Kaneko A, Hayashi N, Tanaka Y, et al. Changes in serum human hepatocyte growth factor levels after transcatheter arterial embolization and partial hepatectomy. Am J Gastroentero/1992; 87: 1014-7

39. Kenworthy P, Dowrick P, Baillie-Johnson H, et al. The presence of scatter factor in patients with metastatic spread to the pleura. Br J Cancer 1992; 66: 243-7. 40. Tomiya T, Tani M, Yamaka S, Hayashi S, Umeda N, Fujiwara K. Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 1992; 103: 1621-4. 41. Taniguchi T, Toi M, Inada K, Imazawa T, Yamamoto Y, Tominaga T. Serum concentrations of hepatocyte growth factor in breast cancer patients. Clin Cancer Res 1995; 1: 1031-4. 42. Hiscox S, Hallett MB, Puntis MCA, Jiang WG. Inhibition of cancer cell motility and invasion by interleukin-12. Clin Exp Metastasis 1995; 13: 396-404. 43. Jiang WG, Hiscox S, Hallett MB, Scott C, Horrobin DF, Puntis MCA. Inhibition of hepatocyte growth factor-induced motility and in vitro invasion of human colon cancer cells by gamma-linolenic acid. Br J Cancer 1995; 71: 744-52.

© 1996 Blackwell Science Ltd, Surgical Oncology, 5: 77-84