Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Summary

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Abstract

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.

The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.

Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.

Introduction

Chemotherapy is still one of the main courses of treatments employed in the clinic for various cancer diseases, and thus vast amount of research is conducted worldwide with the aim to develop new and improved anticancer drugs. Such studies mostly begin at the chemical level, with the design and preparation of compounds, followed by biological evaluation of the cytotoxic properties in vitro. Cell viability may be assessed by various assays that provide information on cellular activity^1-2.

Cisplatin is an example of a platinum complex that is widely used as a chemotherapeutic drug, which is considered an efficient treatmen....

Protocol

1. Preparation of the V (V) complex (Figure 2);¹⁸ conduct steps 1.1-1.4 in a glove-box; if a glove-box is not available, skip to the alternative steps 2 (2.1-2.6).
   1. Dissolve 0.42 mmol of the ligands H₂L in dry THF and add it to a stirring solution of equivalent amounts of VO(OiPr)₃ in THF.
   2. Stir the reaction mixture at room temperature for 15 min, and remove the volatiles under vacuum.
   3. Add cold hexane and remove it under vacuum. A dark purple powder should be obtained in a quantitative yield.
   4. Weigh 8 mg of the obtained compound in an Eppendorf vial. Skip to step 3.
....

Results

The complexes were prepared based on established procedures^18,28 and their purity may be evaluated by NMR and elemental analysis.

The data received from the MTT assay is analyzed to evaluate the cytotoxicity of the compound^18,21. First, subtraction of the blank control absorbance value from all the other values is performed. Second, the THF solvent control value is set to 100% viability, as no growth inhibiting compound was added. All other values are transformed into per.......

Discussion

The method described in this manuscript combines chemical synthesis with biological cell viability assay. As with any biological methods concerning viable cells, it is vital to work in a laminar hood, and maintain sterile conditions, including the use of sterile organic solvents. Also, preparation and storage of hydrolytically unstable metal based drugs should be carried out under inert conditions, for which glove box or Schlenk line techniques should be utilized.

The selection of the techniqu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
Fetal bovine serum (FBS)	Biological Industries	04-007-1A
Hexane AR	Gadot	830122313	Dried using a solvent drying system
HT-29 cell line	ATCC	HTB-38
Isopropanol AR	Gadot	830111370
L-glutamine	Biological Industries	03-020-1B
MTT	Sigma-Aldrich	M5655-1G
Penicillin/streptomycin antibiotics	Biological Industries	03-031-1B
RPMI-1640 with phenol red with L-glutamine	Sigma-Aldrich	R8758
RPMI without phenol red	Biological Industries	01-103-1A
Tetrahydrofuran (THF) AR	Gadot	830156391	dried using a solvent drying system
Ti(OiPr)4	Sigma-Aldrich	205273-500ML	moisture sensitive
Trypsin/EDTA	Biological Industries	03-052-1B
VO(OiPr)3	Sigma-Aldrich	404926-10G	moisture sensitive
Equipment
12-channel pipette 30-300 μl	Thermo Scientific
12-channel pipette 5-50 μl	Finnpipette
75 cm2 flask	NUNC	156472
96-well plate with lid (flat bottom)	NUNC	167008
CO2 Incubator	Binder	APT.line C150
Counter chamber	Marienfeld-Superior	650030
Eppendorf vial	KARTELL
Glove box	M. Braun
Laminar flow hood	ADS LAMINAIRE	OPTIMALE 12
Microplate reader spectrophotometer	Bio-Tek	El-800
Microscope	Nikon	Eclipse TS100
Pipette 20-200 μl	Finnpipette
Pipette 5-50 μl	Finnpipette