Gold(III) compounds are emerging as a new class of metal complexes with outstanding cytotoxic properties and are presently being evaluated as potential antitumor agents. We report here on the solution and electrochemical properties, and the biological behavior of some gold(III) dithiocarbamate derivatives which have been recently proved to be one to 4 orders of magnitude more cytotoxic in vitro than the reference drug (cisplatin) and to be able to overcome to a large extent both intrinsic and acquired resistance to cisplatin itself. Their solution properties have been monitored in order to study their stability under physiological conditions; remarkably, they have shown to undergo complete hydrolysis within 1 h, the metal center remaining in the +3 oxidation state. Their DNA binding properties and ability in hemolyzing red blood cells have been also evaluated. These gold(III) complexes show high reactivity toward some biologically important isolated macromolecules, resulting in a dramatic inhibition of both DNA and RNA synthesis and inducing DNA lesions with a faster kinetics than cisplatin. Nevertheless, they also induce a strong and fast hemolytic effect (compared to cisplatin), suggesting that intracellular DNA might not represent their primary or exclusive biological target.

Gold(III) dithiocarbamate derivatives for the treatment of cancer: solution chemistry, DNA binding, and hemolytic properties.

Dissociated cancer cells are exposed to antineoplastic drugs (5 × 10 4 viable cells/drug for 1 hr or continuously) and cultured for 4 to 6 days in liquid medium. Cells are then stained with Fast green dye, sedimented onto slides with a Cytospin centrifuge, and counterstained with a modified hematoxylin and eosin technique. Dead cells stain with Fast green, and living cells stain with hematoxylin and eosin. Cell kill is calculated as percentage of control based on the relative numbers of living tumor cells, living non-tumor cells, and dead cells. Drug sensitivity could be assayed in 125 of 162 specimens of human neoplasms obtained from malignant effusions (16 of 18), excisional biopsies (31 of 44), needle biopsies (34 of 47), endoscopic biopsies (18 of 23), peripheral blood samples (19 of 20), and bone marrow aspirates (five of seven). The assays were successful (median of ten drugs tested) in: 46 of 64 adenocarcinomas; four of 11 squamous cell carcinomas; five of seven lymphomas; six of seven melanomas; two of four sarcomas; 18 of 20 transitional-cell carcinomas; 14 of 15 small-cell carcinomas; seven of eight myelomas; 12 of 12 chronic lymphocytic leukemias; seven of nine acute leukemias, and four of five "undifferentiated" carcinomas. The assay results demonstrated a strong correlation between the in vitro chemosensitivity of different types of tumors and the known clinical response patterns of these tumors. This assay can be used to determine which specific cells are killed in a heterogeneous cell population. Further work is needed to determine if this assay may be useful in blind screening trials for antineoplastic agents or if it may be of clinical use in predicting response to agents which are not cycle specific.

https://cancerres.aacrjournals.org/content/canres/43/2/749.full.pdf

A Novel Dye Exclusion Method for Testing in Vitro Chemosensitivity of Human Tumors

The following factors must be considered when dye exclusion assays are interpreted. (a) It may require several days for lethally damaged cells to lose their membrane integrity following a cytotoxic insult. (b) During this time, the "surviving" cells may continue to proliferate. (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional "percent viability" expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from two different established tumor cell lines. Results were based upon comparisons of the ratios of "viable" tumor cells to duck erythrocytes on permanent cytocentrifuge slides prepared from the cultures. This novel "ratio" method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All three assays demonstrated a clear dose-effect relationship for most of the drugs tested. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.

https://cancerres.aacrjournals.org/content/canres/43/1/258.full.pdf

Comparison of Dye Exclusion Assays with a Clonogenic Assay in the Determination of Drug-induced Cytotoxicity

Platinum(II) and palladium(II) complexes with dithiocarbamates and amines: synthesis, characterization and cell assay

Monocationic hydrophilic complexes [Cu(thp)4](+) 3 and [Cu(bhpe)2](+) 4 were synthesized by ligand exchange reactions starting from the labile [Cu(CH3CN)4][PF6] precursor in the presence of an excess of the relevant hydrophilic phosphine. Complexes 3 and 4 were tested against a panel of several human tumor cell lines. Complex 3 has been shown to be about 1 order of magnitude more cytotoxic than cisplatin. Chemosensitivity tests performed on cisplatin and multidrug resistance phenotypes suggested that complex 3 acts via a different mechanism of action than the reference drug. Different short-term proliferation assays suggested that lysosomal damage is an early cellular event associated with complex 3 cytotoxicity, probably mediated by an increased production of reactive oxygen species. Cytological stains and flow cytometric analyses indicated that the phosphine copper(I) complex is able to inhibit the growth of tumor cells via G2/M cell cycle arrest and paraptosis accompanied with the loss of mitochondrial transmembrane potential.

In vitro antitumor activity of the water soluble copper(I) complexes bearing the tris(hydroxymethyl)phosphine ligand.

Tumor cells from animals and humans were treated with drugs under tissue culture conditions. Tumor cells from the sensitive L1210 model were studied first. A dose-response curve was derived between drug exposure and subsequent cytotoxicity in L1210. The concentration of drug and duration of exposure were factors critical to the subsequent development of in vitro cytotoxicity. The in vitro dosage which effected 50% leukemic cell death in L1210 cells correlated with reported in vivo drug levels. Other tumor models and human neoplastic cells were studied at this dosage level. A good correlation was noted in these studies between the in vivo responsiveness and the in vitro chemotherapy results in both animals and humans. It was suggested by these results that it may be possible to predict cancericidal drug activity for individual neoplasms by assaying the tumor cells in vitro for drug sensitivity.

A Methodological Approach to the Prediction of Anticancer Drug Effect in Humans

MOPC 104E myeloma cells are brought under host regulation after treatment with cyclophosphamide, 1,3-bis(2-chloroethyl)-1-nitrosourea, or cis-dichlorodiammineplatinum(II). The first indication of this phenomenon is the plateau level of immunoglobulin M(lambda) [IgM(lambda)]. The myeloma recurs more frequently in animals with high plateau levels of IgM(lambda) even when remission is maintained for greater than 200 days. The growth rate of the recurring tumor is altered when compared with the original tumor in the same individual. Different drugs and dosages produce stable myeloma of different sizes. Treatment with cyclophosphamide (10 to 200 mg/kg), or 1,3-bis(2-chloroethyl)-1-nitrosourea (25 mg/kg) gives stable myeloma that produces low plateau levels of IgM(lambda). This myeloma does not show late recurrence. Combination of 1,3-bis(2-chloroethyl)-1-nitrosourea, cyclophosphamide, and cis-dichlorodiammineplatinum(II) in low doses or cis-dichlorodiammineplatinum(II) alone gives a stable myeloma clone(s) which produces IgM(lambda) which plateaus at higher levels, and the myeloma clone recurs relatively late in the life of the animal. These results show that treatment does not lead to the elimination of the dominant myeloma clone. Clonal dominance is, however, broken when the proliferative potential is interrupted by drug treatment. The resulting long stable phase supports the view that the proliferation, the expression of the plasma cell maturation sequences, and the secretion of IgM(lambda) are under normal host regulation. Aging presumably causes a loss of regulatory control permitting clonal expansion and recurrence of the myeloma in animals with high plateau levels of the IgM(lambda). The MOPC 104E myeloma model demonstrates for the first time a conversion of the malignant form to the indolent form as seen for humans.

/pdf/maintenance-of-mopc-104e-myeloma-in-plateau-phase-1kj1u0gkk5.pdf

Maintenance of MOPC 104E myeloma in plateau phase.

Tumor cells from animals and humans were treated with drugs under tissue culture conditions. Tumor cells from the sensitive L1210 model were studied first. A dose-response curve was derived between drug exposure and subsequent cytotoxicity in L1210. The concentration of drug and dura tion of exposure were factors critical to the subsequent development of in vitro cytotoxicity. The in vitro dosage which effected 50% leukemic cell death in L1210 cells correlated with reported in vivo drug levels. Other tumor models and human neoplastic cells were studied at this dosage level. A good correlation was noted in these studies between the in vivo responsiveness and the in vitro chemo therapy results in both animals and humans. It was sug gested by these results that it may be possible to predict cancenicidal drug activity for individual neoplasms by as saying the tumor cells in vitro for drug sensitivity.

A MethodologicalApproachto the Predictionof AnticancerDrugEffect in Humans1

A spontaneous AP positive C3H murine OS was used to determine the effects of various treatment modalities. AP served as a useful circulating biomarker of the in vivo tumor growth. In animals whose tumor was amputated, the elevation of the marker indicated the presence of pulmonary metastases. It was used to establish the time of recurrence after a partially effective chemotherapy or combination modality. In this model, when the neoplasm was surgically excised at 10 days posttransplantation, 70--80% of the mice showed presence of lung metastases. Specific immunotherapy with irradiated tumor cells did not alter the course of the disease. Passively transferred immune allogeneic cells to tumor bearing mice at an effector to target cell ratio of 1:1 in vivo were ineffective. The murine OS model is extremely useful to plan and institute different combinations of treatment modalities and optimize the conditions for an effective treatment in a relatively short time.

Dolores W. Davis

Papers

A Methodological Approach to the Prediction of Anticancer Drug Effect in Humans

Maintenance of MOPC 104E myeloma in plateau phase.

A MethodologicalApproachto the Predictionof AnticancerDrugEffect in Humans1

Tumor immunology of experimental osteosarcoma.

Age related changes in auto-erythrocyte rosettes in the C57BL/6J and NZB/BINJ mice.