Mol. Cells, Vol. 9, No.1 , pp. 31-36
`
`Molecules
`and
`Cells
`
`© Springer-Verlag 1999
`
`Luteinizing Hormone Releasing Hormone-RNase A Conjugates
`Specifically Inhibit the Proliferation of LHRH-Receptor-Positive
`Human Prostate and Breast Tumor Cells
`
`Yong Song GhoT and Chi-Born Chae*
`Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea.
`
`(Received on July 11, 1998)
`
`Human prostate and breast tumor cells produce
`luteinizing hormone-releasing hormone (LHRH)
`receptors on their cell surface even when they have lost
`dependency on sex steroid hormones for growth. To
`investigate whether LHRH can be used as a cell(cid:173)
`binding moiety to deliver toxin molecules into prostate
`and breast tumor cells, LHRH-bovine RNase A
`conjugates were constructed using the chemical cross(cid:173)
`linking method. The treatment of the LHRH receptor(cid:173)
`positive cells such as prostate LNCapFGC and breast
`MCF7 tumor cells with LHRH-RNase A conjugates
`resulted in a dose-dependent inhibition of growth. The
`cytotoxic activities of these conjugates were effectively
`reduced by the presence of exogenous LHRH. Either
`free RNase A or LHRH alone did not affect the
`proliferation of these cells. The LHRH-RNase A
`conjugates did not show cytotoxicity against FRTLS
`and TM4 cells which do not express the LHRH
`receptors. These results suggest that LHRH can be used
`as a cell-binding molecule for the specific delivery of
`toxin molecules into the cells which express LHRH
`receptors on their surface. Thus, a new class of
`biomedicines that act as fusion proteins between LHRH
`and toxins will give us a new avenue for the treatment
`of human prostate and breast cancers, regardless of
`their steroid hormone dependency.
`
`Keywords: Breast; LHRH; Prostate; RNase A; Tumor.
`
`Introduction
`
`An immunotoxin (IT) is a cytotoxic agent that consists of
`a cell-binding moiety and a toxin moiety. The cell-binding
`moiety can be an antibody, a growth factor, or a hormone
`that binds selectively to the cell surface molecules of
`certain cell types. The toxin moiety can be a naturally
`occurring Pseudomonas exotoxin, diphtheria toxin, or
`plant toxin . These immunotoxins can kill cells with
`selectivity in tissue culture and in animal models (Aaron
`and Phillip, 1981 ; Ahmann et ai., 1987). However, the
`toxins derived from bacteria are very immunogenic
`molecules and some immunotoxins are toxic to the liver
`because of the nonspecific uptake by the liver (Bajusz et
`ai., 1989; Beintema et ai., 1984). The use of a member of
`the human ribonuclease A superfamily such as serum
`protein angiogenin may alleviate the problems caused by
`the immunogenicity and liver toxicity (Bond, 1988 ;
`Brinkmann et ai., 1993; Denis and Mahler, 1990; Fekete
`et aI., 1989). When angiogenin was fu sed to the transferrin
`or anti-transferrin receptor monoclonal antibody by
`chemical cross-linking or recombinant methods, these
`immunotoxins exhibit cell type-specific cytotoxic activity
`(Denis and Mahler, 1990; Fekete et aI. , 1989). In contrast,
`angiogenin alone does not have any cytotoxicity at
`appropriate concentrations.
`LHRH is a decapeptide hormone released by the
`hypothalamus and acts on gonadotropes at the anterior
`pituitary to stimulate the release of the luteinizing hormone
`(LH) and follicle-stimulating hormone (FSH). Potent
`agonists or antagonists of LHRH have been used for the
`
`* To whom correspondence should be addressed.
`Tel: 82-562-279-2125; Fax: 82-562-279-2199
`E-mail: cbchae@postech.ac.kr
`
`t Present address: Cell Biology Section, National Institute of
`Dental Research, MIH, Bethesda, MD 20892, USA.
`
`Abbreviations : EDC , l-ethyl-3-(3-dimethylamino propyl)
`carbodiimide·HCl; FSH, follicle-stimulating hormone; IT,
`immunotoxin ; LH, luteinizing hormone; LHRH, luteinizing
`hormone-releasing hormone.
`
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`32
`
`Killing Tumor Cells by LHRH-RNase A
`
`treatment of sex steroid hormone-dependent tumors such
`as prostate and breast cancers (Isaacs and Coffey, 1981).
`The suppression of gonadal steroid secretion by hormonal
`therapy with these agonists and antagonists retards the
`growth of these hormone-dependent tumors . However,
`these hormone analogs may also act directly on the tumors.
`Human prostate and breast cancer cells produce LHRH
`receptors on their cell surface even when they have lost
`their dependency on sex steroid hormones for growth, and
`LHRH agonists or antagonists can inhibit the growth of
`some breast cancer cell lines (Jennes et a!., 1988; Miller et
`al., 1985 ; Newton et al., 1994). Highly potent
`metallopeptide analogs of LHRH show selective
`cytotoxicity against human prostate and breast cancer cell
`lines in vitro, and LHRH analogs containing cytotoxic
`radicals inhibit the growth of estrogen-independent MXT
`mouse mammary carcinoma cells in vivo (Newton et a!.,
`1996; Pai et a!., 1992). The LHRH analogs containing
`cytotoxic agents may be useful for the treatment of human
`prostate and breast cancer, especially for the hormone(cid:173)
`independent tumors because hormonal therapy does not
`prevent the growth of hormone-independent cells (Pai et
`al. , 1990). These results implicate that LHRH can be used
`as a cell-binding moiety to deliver toxin molecules such as
`ribonuclease into prostate and breast tumor cells regardless
`of their steroid hormone dependency for growth.
`To investigate this possibility, LHRH-bovine RNase A
`conjugates were constructed using the chemical cross(cid:173)
`linking method. According to our preliminary experiments,
`these conjugates show cell type-specific cytotoxicity, and
`the cytotoxicity of these conjugates is abolished by the
`presence of excess LHRH. The res ults suggest that
`decapeptide LHRH can be successfully used as a cell(cid:173)
`binding molecule for the specific delivery of toxin
`molecules into the cells which express LHRH receptors on
`their surface. Thus, construction of the fusion protein
`between LHRH and a member of the human ribonuclease
`superfamily such as pancreatic RNase A, angiogenin and
`eosinoprul-derived neurotoxin will give us a new avenue to
`treat the human prostate and breast cancers regardless of
`their steroid hormone dependency.
`
`Materials and Methods
`
`Chemical conjugation of LHRH and RNase A Bovine
`pancreatic RNase A (2 mg, Sigma) and LHRH free acid (5 mg,
`Bachem California) were dissolved together in 0.8 mJ of water,
`and the pH of the mixture was adjusted to 7-8. Next, l-ethyl-3-
`(3 -dimethylamino propyl) carbodiimide·HCI (100 mg, EOC,
`Pierce) freshly dissolved in 0.2 ml of water, was added . The
`reaction was permitted to proceed with gentle agitation at room
`temperature for 2 h and terminated by gel filtration on a POlO
`column (Pharmacia) to remove the unreacted EDC and LHRH.
`The sample was further dialyzed against water for 24 h with three
`changes. The resultin g LHRH-RNase A conjugates were
`
`aliquoted and stored at -70°C until use. The amount of
`conjugated protein was quantified by Bio-Rad protein assay
`reagents (Bio-Rad Laboratories) using bovine serum albumin as
`a standard.
`
`Assay of ribonucJeolytic activity The ribonucleolytic activity
`was determined in a reaction mixture (0.1 ml) containing 4 mg/ml
`of yeast tRNA (type X, Sigma), 10 ~ of PMSF, 0.1 mg/ml of
`human serum albumin (Calbiochem), 33 mM HEPES/NaOH pH
`7.5, 33 mM NaCl , and appropriate concentrations of RNase A or
`LHRH-RNase A conjugates. After 15 min incubation at 37°C, the
`reaction was termi nated by the addition of 0.24 ml of 3.4% ice(cid:173)
`cold perchloric acid. The samples were kept on ice for 10 min and
`then centrifuged for 10 min at 4°C. Absorbance of the supernatant
`was determined at 260 nm (Ramakrishnan et al., 1992). Tbe
`activity of LHRH-RNase A conjugates was compared with that of
`non-conjugated RNase A. All experiments were carried out in
`duplicates.
`
`Cell culture LNCap·FGC human prostate adenocarcinoma
`cells (ATCC CRL 1740) were grown in -RPMI 1640 with 10%
`fetal bovine serum. MCF7 human breast adenocarcinoma cells
`(ATCC HTB 22) were cultured in Eagle's MEM with 10% fetal
`bovine serum. TM3 mouse Leydig cells (ATCC CRL 1714) and
`TM4 mouse Sertoli cells (ATCC CRL 1715) were grown in a 1: 1
`mixture of Ham's F12 medium with Oulbecco's modified Eagle's
`medium containing 5% horse serum and 2.5% fetal bovine serum.
`FRTL-5 rat tbyroid cells (ATCC CRL 8305) were cultured in
`Coon 's modified Ham's F12 medium with 5% bovine calf serum.
`Cells were subcultured after trypsinization , and the medium was
`changed every 2-3 d.
`
`Cytotoxicity assay Each well of a tissue culture plate (96 well,
`Nunc) was plated with 104 cells. After incubation at 37°C for 2 d,
`100 III of varying amounts of LHRH-RNase A conjugates, LHRH
`or RNase A samples in culture medium was added to each well.
`After incubation for two additional days, the number of viable
`ce ll s was determined usi ng Cell Titer 96 Non-Radioactive
`Proliferation assay kit (Promega) according to the manufacturer's
`instructions. Each experiment was carried out in triplicates, and
`an average value was used to determine percent viability.
`A time course of the action of LHRH-RNase A conjugates
`against LNCapFGC and MCF7 cells was investigated as follows .
`Cells were placed in a 96-well tissue culture plate at 103 cells per
`well and incubated for 2 d. The cells were grown for 2, 5, and 9
`additional days in the presence of 111M of LHRH-RNase A
`conjugates.
`
`Results
`
`Preparation of LHRH-RNase A conjugates Natural
`LHRH does not have any free carboxyl or amino groups
`(pGl u- His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH 2) .
`Therefore, bovine pancreatic ribonuclease A (RNase A)
`was coupled to LHRH free acid which contains a Gly-free
`carboxyl group at the C-terminus instead of Gly-arnide
`using the heterobifunctional crosslinking agent EDC. Due
`to the difficulty of separation of LHRH-RNase A
`
`IMMUNOGE 2156, pg. 2
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`

`Yong Song Gbo & Chi-Born Cbae
`
`33
`
`conjugates from RNase, RNase A was reacted with a
`3S-fold molar excess of LHRR free acid. The unreacted
`LHRH and EDC were separated from the conj ugates by gel
`fi ltration on a PD 10 desalting column. Successful
`conjugation between the reactants was confirmed by SDS(cid:173)
`PAGE and Western blotting using monoclonal antiLHRH
`antibody (data not shown). The ribonucleolytic activity of
`LHRH-RNase A was lO-fold lower than that of RNase A.
`This reduction of enzymatic activity of the conjugates may
`be due to the modification of some lysine at the active
`center of RNase A (Rodriguez et aI., 1993).
`
`The cell type-specific cytotoxic effect of LHRH-RNase
`A conjugates To investigate whether LHRH-RNase A
`conjugates can inhibit proliferation of the cells which
`retain LHRH receptors on their surface, human prostate
`adenocarcinoma LNCapFGC cells and human breast
`adenocarcinoma MCF7 cells were treated with varying
`amounts of conjugates for 2 d. As shown in Figs. 1 and 2,
`the conj ugate s inhibit the proliferation of these
`cell s in a concentration -dependent manner, and the
`concentrations of conj ugates giving a SO% inhibition of
`cell proliferation were found to be -O.S )lM and -0.7 )lM
`for LNCapFGC and MCF7 cells, respectively. However,
`RNase A and LHRH alone do not affect the growth of both
`cells at up to 10 )lM. Therefore, the chemical attachment of
`LHRH to RNase A is needed for the cytotoxicity of these
`conjugates.
`To demonstrate that the cytotoxicity of LHRH-RNase A
`conj ugates was due to the binding to the LHRH receptor,
`two types of experiments were carried out. First, the
`cytotoxic activity of varying concentrations of conjugates
`against LNCapFGC and MCF7 cells was investigated in
`the prese nce of 10 nM of LHRH. Exogenous LHRH
`effectively reduces the cytotoxicity of LHRH-RNase A
`conjugates against these cells (Figs. 1 and 2). Secondly, we
`investigated whether LHRH-RNase A conjugates could be
`delivered into specific cells which express LHRH receptors
`on their surface. The conj ugates had no cytotoxic activities
`against LHRH-receptor-negative Sertoli TM4 cells and
`thyroid FRTLS cells (Fig. 3). However, the proliferation of
`Leydig TM3 cells which express LHRH receptors on their
`cell surface was also inhibited by the presence of
`conjugates.
`
`__ LHRH-RNase A
`-0-- RNase A
`_____ LHRH
`---'V- LHRH-RNase A + LHRH (10 nM)
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`<a
`>
`.~
`
`:::l
`(/)
`~
`
`-10
`
`-9
`
`-8
`
`-7
`
`-6
`
`-5
`
`-4
`
`Log [Concent rat ion]
`
`Fig_ L Cytotoxicity of LHRH-RNase A conju gates against
`LNCapFGC prostate cancer cells. LNCapFGC cells were treated
`with varying concentrations of LHRH-RNase A, RNase A and
`LHRH for 2 d and percentage viability was determined as
`described in Materials and Methods. The cytotoxic activity of
`varying concentrations of LHRH-RNase A conj ugates against
`LNCapFGC cells was also investigated in the presence of 10 nM
`of LHRH.
`
`- - LHRH-RNase A
`-0-- RNaseA
`_____ LHRH
`---'V- LHRH-RNase A + LHRH (10nM)
`
`T
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`<a
`>
`.~
`
`:::l
`(/)
`~
`
`Prolonged inhibition of cell proliferation by LHRH(cid:173)
`RNase A conjugates We investigated a time course of
`the cytotoxic activity of LHRH-RNase A conj ugates
`against LNCapFGC and MCF7 cells by growing the cells
`for 2, S, and 9 d in the presence of conj ugates. The
`inhibition of the growth of LNCapFGC and MCF7 cells
`continued for 9 d. This effect is not due to the possible
`deterioration of culture medium since the control cells
`grew continuously in the absence of conj ugates for 9 d in
`the culture medium without replacement (Fig. 4).
`
`·10
`
`-9
`
`-8
`
`-7
`
`-6
`
`-5
`
`-4
`
`Log [Concentration]
`
`Fig. 2. Cytotoxicity of LHRH-RNase A conjugates against
`MCF7 breast cancer cells. MCF7 cells grown in culture were
`treated with varying concentrations of LHRH-RNase A, RNase A
`and LHRH for 2 d and percentage viability was determined as
`described in Materials and Methods. The cytotoxic activity of
`varying concentrations of LHRH-RNase A conjugates against
`MCF7 cells was also investigated in the presence of 10 nM of
`LHRH.
`
`IMMUNOGE 2156, pg. 3
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`IPR2014-00676
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`

`

`34
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`IV
`>
`.~
`
`:::l en
`<f!.
`
`·10
`
`·9
`
`·8
`
`·7
`
`·6
`
`·5
`
`-4
`
`Log [Concentratio n]
`
`Fig. 3. Cell type-specific cytotoxic activities of LHRH-RNase A
`conjugates . Leydig TM3 , Sertoli TM4, and thyroid FRTL5 cells
`grown in culture were treated with varying amounts of LHRH(cid:173)
`RN ase A conjugates for 2 d and percentage viability was
`determined as described in Materials and Methods.
`
`---- LNCapFGC
`-0- LNCapFGC + LHRH-RNase A
`--.- MCF7
`-<r- MCF7 + LHRH-RNase A
`
`T
`
`0 .... OJ) <
`
`2.0
`
`1.6
`
`1.2
`
`.8
`
`.4
`
`0.0
`
`2
`
`4
`
`8
`
`10
`
`Incubation Days
`
`Fig. 4. Time course of the action of LHRH-RNase A conjugates.
`LNCapFGC and MCF7 cells were treated with 1 ~M of LHRH(cid:173)
`RNase A for 2, 5, and 9 d and viable cells were determined as
`described in Material s and Methods. For control experiments,
`LNCapFGC and MCF7 cells were grown in the absence of
`LHRH-RNase A conjugates.
`
`Killing Tumor Cells by LHRH-RNase A
`
`---- TMJ
`-0- TM4
`--.- FRTL5
`
`Discussion
`
`Prostate and breast cancers are the major cancers leading to
`death in American men and women, respectively. The
`functions of the prostate and breast are regulated by steroid
`hormones such as androgen and estrogen, respectively.
`Furthermore, these sex hormones are necessary for the
`growth of hormone-dependent human prostate and breast
`tumors. Available options for the treatment of prostate and
`breast cancers are surgical orchiectomy, radiation therapy,
`hormone treatment and cytotoxic chemotherapy.
`Administration of analogs of the natural decapeptide
`LHRH stimulates the secretion of LH and FSH by
`gonadotropes. When chronic administration is continued,
`LH and FSH secretion is decreased by desensitization of
`the LHRH receptors in the gonadotropes, leading to a
`decrease of circulating steroid hormones and thus
`inhibiting the growth of prostate and breast cancers
`(FitzGerald and Gordon, 1992; Isaacs et al., 1981).
`However, surgical and hormonal combination therapy to
`eliminate the testicular testosterone or the ovarian estrogen
`leaves an opportunity for the adrenal androgen and
`estrogen secreted by adrenal glands to stimulate the growth
`of prostate and breast cancers. Furthermore, hormone(cid:173)
`independent prostate cancer (androgen-independent) and
`breast cancer (estrogen-independent) can still grow after
`these types of combinational treatments have been applied.
`In the case of cytotoxic chemotherapy, the standard
`cytotoxic agents that are normally used against other
`cancers primarily act against rapidly growing tumor cells,
`but prostate cancer is not a rapidly growing tumor. It is not
`clear whether some combination of chemotherapeutic
`agents which is effective for the treatment of hormone(cid:173)
`independent tumors will be found. It seems that it is time
`to try some new avenues. In this sense, the hormone-toxin
`approach is intriguing.
`The purpose for the construction of LHRH-RNase A
`conjugates by chemical conjugation was to evaluate the
`potential of these conjugates to kill human prostate and
`breast cancer cells which express LHRH-receptors. We
`found that the LHRH-RNase A conjugates can selectively
`kill the LHRH receptor-positive cells such as Leydig TM3 ,
`prostate LNCapFGC and breast MCF7 tumor cells but not
`FRTLS and TM4 cells which do not express the LHRH
`receptors. Extracellular RNase A or LHRH alone is not
`cytotoxic toward LNCapFGC and MCF7 cells. Also, the
`cytotoxic activities of these conjugates can be effectively
`reduced by the presence of exogenous LHRH. These
`results clearly indicate that LHRH-RNase A conjugates can
`kill the specific cell types which express the LHRH(cid:173)
`receptors and that the cell type-specific cytotoxic activities
`are due to the delivery of RNase A to the cytosol via
`LHRH receptor-mediated endocytosis.
`The relatively high ICso value of these conjugates might
`be due to the fact that the chemically linked LHRH-RNase
`
`IMMUNOGE 2156, pg. 4
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`IPR2014-00676
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`

`Yong Song Gho & Chi-Born Chae
`
`35
`
`A conjugates are heterogeneous and that the ribonuclease
`activity of the conjugate was 10-fold lower than that of
`natural RNase A . According to prev ious reports, the
`recombinant fusion protein between ribonuclease and the
`monoclonal antibody to the tran sferrin receptor was a
`thousand times as potent as the chemically linked hybrid
`(Bond, 1988; Denis and Mahler, 1990). Thus, construction
`of the fusion protein between LHRH and a member of the
`human ribonuclease superfamily such as pancreatic RNase
`A , angiogenin and eosinophil-derived neurotoxin
`eliminates the heterogeneity of chemically cross-linked
`LHRH-RNase A conjugates and may increase the cell type(cid:173)
`s pec ifi c cytotoxicity. Although seve ral kind s of
`immunotoxins which show the cytotoxic activity against
`human prostate and breast cancer in vitro and in vivo have
`been developed as a fusion protein between the specific
`antibody for target cells and bacterial or plant toxin as a
`toxin moiety (Rybak et aI. , 1991 ; 1992; Sharoni et aI.,
`1989), recombinant LHRH-RNase toxins may have several
`potential therapeutic advantages to immunotoxins for the
`following reasons. In contrast to previously developed
`immunotoxins, these new biomedicines may not cause
`immunogenesis and liver toxicity. These LHRH-RNase
`toxins may penetrate easily into the solid tumor due to
`their small size. Both Leydig cells in the testis and
`Granulosa cel ls in the ovary al so express the LHRH(cid:173)
`receptor on their surfaces (Isaacs et aI., 1981). These cells
`are major sources of androgen and estrogen which are
`needed for the growth of prostate and breast cancer cells,
`respectively. Thus, LHRH-RNase toxins may not only
`selectively kill the prostate and breast cancer cells, but also
`effectively eliminate the circulating androgen and ovarian
`estrogen by eliminating either the Leydig cells in men, or
`the Granulosa cells in women. LHRH receptors are also
`present in the central nervous system (Szepeshazi et aI.,
`1992). However, LHRH agonist has been used to treat
`human prostate and breast cancers and LHRH-cytotoxic
`radicals produce significant inhibition of tumor growth
`without visible effect on the brain function (Pai et aI. ,
`1992; Vitetta et aI., 1983). This means that the circulating
`LHRH agonist and LHRH-toxin may not penetrate the
`brain-blood barrier. Similarly, LHRH-RNase toxins may
`not reach the brain nor cause any side effect on the brain
`function.
`In conclusion, LHRH-RNase A conjugates which may
`not have immunogenic problems or liver toxicity will give
`us a new avenue for the treatment of human prostate and
`breast cancers regardless of their steroid hormone(cid:173)
`dependency. In addition, its combination with surgical and
`honnonal therapy which have been used for the treatment
`of these cancers may increase their therapeutic potency.
`
`Acknowledgments This work was supported in part by
`Pohang University of Science and Technology and the Korea
`Green Cross, Inc.
`
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