Antitumour and Immunomodulatory Effects of Cu(II) Complexes of Thiobenzyhdrazide

Thiiobenzyhdrazide (Htbh) and its Cu(II) complexes, [ Cu ( Htbh ) 2 Cl 2 ] and [ Cu ( tbh ) 2 ] were synthesized and characterized by various physicochemical studies. In vivo and in vitro antitumour activity of Htbh, [ Cu ( Htbh ) 2 Cl 2 ] and [ Cu ( tbh ) 2 ] has been tested. LD 50 values were calculated for all the three compounds. It was observed that the antitumour effect of [ Cu ( Htbh ) 2 Cl 2 ] is maximum. Light microscopic study of the treated tumour mass demonstrated that certain cellular degradation, such as disappearance of mitotic figures, loss in cellular compactness, distortion of nucleus and disruption of cytoplasmic boundaries, takes place in the tumour region of complex treated mice. Further, tumour bearing mice administered with Cu(II) complexes showed reversal of tumour growth associated induction of apoptosis in lymphocytes.


INTRODUCTION
During the past decade atter the successful achievement of cis-dichlorodiammine platinum(ll) (cisplatin), a number of derivatives of thiosemicarbazone, such as 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) copper complexes (Cu-KTS), have been found to exhibit antitumour activity/1,2/by binding with DNA/3/. In addition, the copper complexes of thiosemicarbazide inactivate lambda phage infectivity and transaction by lambda DNA/4/, and inhibit the activity of RNA dependent DNA polymerase of Rous sarcoma virus/5/.
Thiosemicarbazones of l-formylisoquinoline and 2-formylpyridine and their derivatives were also demonstrated as effective against animal tumour /6,7/ at the molecular level by inhibiting the enzyme ribonucleoside diphosphate reductase and synthesis of DNA/8-10/. Many chemotherapeutic agents have also been reported to possess immunomodulatory properties/11,12/. However, the immunomodulatory potential of the metal complexes with antitumour activity has not been explored adequately. Although thiohydrazides are structurally quite similar to thiosemicarbazides, scarcity of work on the antineoplastic and immunomodulatory activity of transition metal complexes of thiohydrazides prompted us to study the activity of l'ol. 9, No,s'. I-Z 2002 Antitumour and lmmmlomodulatory Ej]8cts of Cu(ll) Complexes transition metal complexes of thiobenzhydrazide (C6HsC(S)NHNH2). In the present investigation we report the synthesis, characterization and antitumour activity of Cu(ll) complexes of thiobenzhydrazide (Htbh). Further, we also investigated the immunomodulatory action of these complexes on Dalton's Lymphoma (DL) bearing host.

MATERIALS AND METHODS
Elemental analyses, magnetic susceptibility, infrared, electronic and ESR spectra of the complexes were recorded as described elsewhere/12/.

Synthesis of ligand
Carboxymethyldithiobenzoate (2.0g) was dissolved in 1N-NaOH (10 mL, equi) and 10 mL water. The solution was cooled in ice and hydrazine hydrate (0.44g, 2 equi) was added. The thiobenzhydrazide separated immediately and the orange colour of the solution disappeared. A few drops of 1N-HC1 were added to the above solution to bring it to pH 5-6 and the mixture was kept in ice for one hour. The product thus obtained was filtered off, washed with water and dried in vacuo. The crude product was recrystallized from hot ethanol (rap 73-75 C) /13/ (Fig. 1 [Cu(Htbh)Cl] was prepared by adding an ethanol solution of copper(ll) chloride, containing a few drops of dil. HCI to the ethanol solution of the thiobenzhydrazide in a 1:2 molar ratio. The complex which precipitated immediately, was filtered by suction, washed with ethanol and dried in vacuo. [Cu(tbh)], on the other hand, was prepared by adding an ethanol solution of the ligand (thiobenzhydrazide) to an aqueous solution of copper(ll) acetate in a 1:2 molar ratio in the presence of sodium acetate (0.5g) and digesting the reaction mixture for 30-60 min. The complex thus obtained was filtered by suction, washed successively with water, ethanol and finally with ether, and dried in vacuo (Fig. 2).  (Table I). On alternate days, up to 4 weeks tumour bearing animals were weighed to detemaine the change in body weight. The acute toxicities of the complexes were determined after 48h of compound injection in tumour bearing mice. LDs0 values obtained are given in Table i.

Evaluation of antitumour activity
The therapeutic effectiveness of Htbh, [Cu(tbh)2] and [Cu(Htbh)2Cl] against DL bearing mice was assessed from the mean survival time of the treated animals (excluding tumour free survivors) divided by mean life span of untreated mice multiplied by 100, giving the T/C percentage. A T/C value of 115 indicates significant activity, wherewas a value for T/C > 125 indicates that the complex is worthy of testing in other tumour systems.

Histological study
Animals from both control and treated batches were killed at 2-day intervals up to 6 days. Tumour tissue with liver, kidney and spleen was fixed in Bouin's fluid (aqueous), dehydrated, kept in cedar wood soil for 3 days and embedded in paraffin. Paraffin sections of 5 lam were cut, stained in Ehrlich's hematoxylin eosin stain, dehydrated, cleared in xylene, and mounted in DPX. Slides were studied under the light microscope.
All other biological studies were carried out as described elsewhere/12/. However, thioamide band-IV [mainly due to v(C=S)] undergoes a negative shif of 40 cm " in [Cu(Htbh)2CI2], indicating bonding through 'thione' sulfur. This band is, however, found to be absent in [Cu(tbh)2] and in place of this a new band appears at 720 cm"t, due to v(C-S), which indicates bonding through 'thiolato' sulfur/13/ (Fig. 2).

ANTITUMOUR STUDIES
To study the antitumour activity, the ligand and its Cu(ll) complexes were administered (60 and 120 mg/kg of mouse weight) to Dalton's Lymphoma (DL) bearing mice. Toxic doses were evaluated in terms of HNTD, TDH and LDs0 (Table 1). Maximum in vivo antitumour activity was found for [Cu(Htbh)2CI2]. The enhanced activity of the chloro complex as compared to [Cu(tbh)2] may be attributed to the presence of labile chlorine, as is observed in the case of cisplatin. Also, the number of DL bearing mice administered with [Cu(Htbh)2CI2] surviving for more than 3 months was found to be maximum ( Table 2). We checked the life prolongation effect in DL bearing mice administered with phosphate buffer saline (PBS) alone or containing ligand or its Cu(ll) complexes. As shown in Table 2, maximum % T/C was found for [Cu(Htbh)2CI2] followed by [Cu(tbh)2], whereas the ligand alone failed to increase significantly the life span of tumour bearing mice. Increase in the value of % T/C indicates a prolongation of the life of tumour bearing mice and suggests that such an effect could result either from the direct cytotoxic/cytostatic action of the complexes on tumour cells or from the activation of certain host derived antitumour defense mechanism(s). Therefore, in the next part of the study we carried out investigations to understand the mechanism(s) underlying the prolongation of the life span of tumour bearing mice treated with Cu(ll) complexes.
Part of the evidence comes from the experiments in which tumour cells, incubated in the presence or absence of metal complexes, were checked for their effect on viability by MTT assay, in which 3,(4,5- indicating an inhibition of the SDH activity. These results thus indicate a possible decline of the overall metabolic activity of the tumour cells with a concomitant inhibition of the activity of the enzymes involved in halting the process of respiration. Although the metal complexes showed cytostatic effects on the tumour cells in vitro, these results do not necessarily indicate if these cells are actually killed by the direct action of the metal complexes. To check this in the next part of the investigation we studied the effect of the metal complexes on tumour cell killing to [Cu(Htbh)zC12] was found to be most effective in the induction of tumour cell apoptosis (Fig. 3a). The mechanism of the induction of apoptosis remains poorly understood and is thought to be dependent on multiple mechanism(s) ultimately culminating in the activation of DNA cleaving endonucleases/15/. Indeed, results presented in Fig. 3b show that [Cu(Htbh)2C12] and [Cu(tbh)] cause an increase in the percentage of specific DNA fragmentation, a hallmark feature of apoptosis, indicating that these metal complexes may induce apoptosis culminating in the activation of endonucleases causing DNA fragmentation. Light microscopic studies reveal that untreated DL cells show compact cellular organization, in which the cells have an oval shaped large nucleus with a single prominent nucleolus. The cytoplasma is well developed and chromatin material is prominent. Cells in the various stages of mitotic division could be seen. A few leucocytes and lymphocytes were observed. The treated animals revealed the loss of cellular compactness of the tumour mass and cytoplasm appears to be distorted with vesicle appearances. The cell walls of rnost of the cells were ruptured, nuclei became translucent and lost their shapes. The nucleoli material became more Antitumour and Immunomodulatory ffects f Cu(ll) Complexes condensed. The high dose treatment was more effective and cell cytoplasm and the cytoplasmic boundaries between the cells were disrupted. The nucleus became more translucent and nuclear material was in the form of condensed granules. In the case of [Ctl(Htbh)2Cl2] a more effective result was obtained, in which the completely destroyed cytoplasmic mass was prominent. A large number of leucocytes, lymphocytes and macrophages appeared. Only a few mitotic figures were observed after ten doses of treatment. The results reported in this study showed that [Cu(Htbh)zClz] has a strong antitumour effect and also plays an important role in the tumour regression. The appearance of lymphocytes and macrophages in the tumour mass after the treatment with metal complexes also suggests that probably the host's immune defence mechanism is increased. This prompted us to investigate if the administration of metal complexes could reverse tumour growth associated induction of apoptosis in various hematopoietic cells. For this, DL bearing mice were administered with Cu(II) complexes, and the percentage of apoptotic thymocyte, splenocyte and bone marrow cells were enumerated. As shown in Fig. 4a, administration of metal complexes in tumour bearing mice resulted in the inhibition of tumour associated apoptosis of thymocyte, splenocyte and bone marrow cells. Similar results were obtained for percentage DNA fragmentation as well (Fig. 4b). The reversal of tumour growth associated inducation of apoptosis of hematopoietic cells by metal complexes is predicted to be due to reduction of tumour load resulting from the cytotoxic effect of metal complexes on tumour cells, leading to a decrease in the tumour associated concentration of apoptotic factors and by the direct protective effect of metal complexes on the hematopoietic cells. Although not very clear, the probability of the latter could be due to the fact that metal complexes can bind to DNA and several proteins in cells, which could result in the protective effect.
The present study shows that the Cu(ll) complexes of thiobenzhydrazide are not only active antitumour agents, but are also immunopotentiating agents, on account of their ability to reverse tumour associated immunosuppression. The importance of such work lies in the possibility that the new complexes might be more efficacious drugs for therapeutic use against tumours in view of their dual mechanism of action.