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 treatment mainly for testicular and ovarian cancers^3-4. However, its narrow activity range and severe side effects trigger studies of other potent transition metal complexes^5-8. Among others, titanium (IV) and vanadium (V) complexes showed promising results of high activity and reduced toxicity^9-16. Ti (IV) complexes were the first to enter clinical trials after cisplatin due to these properties; however, they have failed the trials due to formulation difficulties and hydrolytic instability. There is thus a current need to develop improved derivatives of these metal complexes that may combine high anticancer activity with water resistance^15,17-21.

A challenge in the preparation of Ti (IV) and V (V) complexes refers to the hydrolytic instability of the precursor reagents; therefore, inert atmosphere should be maintained. The preparation of Ti (IV) and V (V) compounds is conducted under N₂ or Ar conditions in a glove box or using Schlenk line techniques.

One common method for evaluation of the anti-cancer activity is based on the MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) assay. This assay is a colorimetric viability assay that was presented in 1983 by Mosmann²². It is extremely well studied and characterized, and it is considered highly efficient when evaluating the effectiveness of new cytotoxic compounds due to its precision, rapidity, and its capability to be applied on variety of cell lines. This viability assay is based on the color change of the MTT molecule when it is exposed to viable cells. Measurement of the absorbance, which is proportional to the number of viable cells, and comparison to untreated controls, enables assessment of the cell growth inhibition capabilities of the compound tested.

The MTT colorimetric assay is conducted in a 96-well plate format²³. The cells may require preincubation in the wells before the addition of the tested drug. The preincubation times may vary from 0-24 hr according to the cell line properties. Cells are usually exposed to the drug for 24-96 hr depending on the drug activity. MTT solution is then added to the treated cells, where the yellow MTT is reduced to purple formazan by a variety of mitochondrial and cytosolic enzymes that are operational in viable cells (Figure 1)²⁴. The MTT molecule is not reduced by dead cells or red blood cells (metabolically inactive cells), spleen cells (resting cells) and concanavalin A-stimulated lymphocytes (activated cells)²². After 3-4 hr of incubation with MTT the formazan precipitates. The formation of the formazan begins after 0.5 hr of incubation but for optimal results it is best to expose the cells to MTT for at least 3 hr²². Consequently, the growing medium is removed and the formazan is dissolved in an organic solvent, preferably isopropanol²⁵, although DMSO can also be used²⁶. The elimination of the medium is crucial for achieving accurate results since phenol red, which is widely common in growth medium, and precipitating proteins can interfere with the absorbance measurement²⁵. When the formazan solution reaches homogeneous, the absorbance of the solution is measured using a microplate reader spectrophotometer. The absorbance at 550 nm is directly proportional to the number of cells in range of 200-50,000 cells per well, and thus very small amounts of cells can be detected²². The absorbance indicates the amount of viable cells that remained after treatment with the drug, and is compared to the absorbance of control cells that were not exposed to the drug. Analysis of the results by proper software provides the IC₅₀ (inhibition concentration; 50%) values and their statistical errors based on several repetitions of the measurement.

The MTT assay is widely common in cytotoxicity studies for screening new anticancer compounds, due to its accuracy and relative simplicity. However, when using the MTT assay, which is depended on enzymatic reaction, one must consider that various enzyme inhibitors can affect the reduction of MTT and lead to false results²⁷. In addition, the MTT assay does not provide any information on the molecular mechanism of the cytotoxic activity of the drug².

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.
2. Prepare the V (V) complex using a Schlenk line.
   1. Dissolve 0.42 mmol of the ligand H₂L in dry THF in a dry Schlenk flask with a septum cap, attach to a Schlenk line and apply alternating vacuum/inert gas (N₂ or Ar) environments; it is recommended to apply three such rounds and wait 2-5 min on the vacuum stages.
   2. Add dry THF via syringe through the septum cap.
   3. Add equivalent amounts of VO(OiPr)₃ in a THF solution, which had been prepared similarly by hooking a proper dry Schlenk flask to the line, applying an inert environment and adding the reagents through a syringe from the vanadium solution to that of the ligand.
   4. Stir the reaction mixture at room temperature for 15 min and remove the volatiles under vacuum; if there is a concern of unstable products, perform Schlenk type distillation of the volatiles under inert atmosphere; use inert gas flow to attach the required glassware that had been predried.
   5. Add cold hexane and remove it under vacuum. A dark purple powder should be obtained in a quantitative yield.
   6. Weigh 8 mg of the obtained compound in an Eppendorf vial.
3. Preparation of the Ti (IV) complex (Figure 2);²⁸ conduct steps 3.1-3.3 in a glove-box; if a glove-box is not available, adjust the alternative steps discussed for the vanadium analogue (step 2) that employ a Schlenk line instead.
   1. Dissolve 0.35 mmol of the ligand H₂L in dry THF and add it to a stirring solution of equivalent amounts of Ti(OiPr)₄ in THF.
   2. Stir the reaction mixture at room temperature for 2 hr. Remove the volatiles under vacuum to give a yellow product in a quantitative yield.
   3. Weigh 8 mg of the obtained compound in Eppendorf vial.
4. Preparation of HT-29 cells 96-well plate
   1. Culture HT-29 cells in a 75 cm² flask with RPMI-1640 medium that contains 1% penicillin/streptomycin antibiotics, 1% L-glutamine, and 10% fetal bovine serum (FBS), at 37 °C with 5% CO₂.
   2. Remove the medium when the HT-29 cells reach maximal confluency (90-100%, every 3-4 days without subculture). Wash with 1 ml of trypsin (0.25%)/EDTA (0.05%) solution and remove it.
   3. Add 1 ml of trypsin (0.25%)/EDTA (0.05%) solution and incubate for 5 min.
   4. Detach the HT-29 cells from the flask and add 10 ml of the medium to disable the trypsin activity. Transfer 5 ml of the cells mixture to a tube.
   5. Use pipette to transfer a few drops of the cells mixture into counter chamber. The counter chamber includes 5 x 5 squares.
   6. Count the cells in 5 representative squares using a microscope: the 4 squares in the corners and the one that is in the middle.
   7. Calculate the estimated amount of cells present. Sum the cells in the 5 representative squares and divide by 20.
   8. Divide 0.6 by the result of step 4.7. The received number is the amount of cell mixture required per plate. This calculation refers to a plate that contains 0.6 x 10⁶ cells (9,000 cells per well).
   9. Use pipette to transfer the amount of cell mixture required per plate (as calculated in section 4.8) and add 13.2 ml of the medium (200 μl per well × 66 wells).
   10. Add the mixture to 96-well plate by 11-channel pipette (200 μl per well, 6 lines, 66 wells in total). One well of each line should remain empty for blank control.
   11. Incubate the plate at 37 °C with 5% CO₂ for 24 hr to allow the cells to attach to the plate.
5. Insertion of the compounds
   1. Add 200 μl (or different amount according to the desired concentration range) of dry THF to 8 mg of each compound weighed as described in step 1.4 (or 2.6) or 3.3. Weigh 8 mg of the ligand H₂L, too.
   2. Dilute each compound solution further by adding 60 μl of THF in 9 different Eppendorf vials.
   3. Transfer with a pipette 60 μl from the mixture in section 5.1 to the first Eppendorf vial, resuspend and transfer 60 μl to the next vial, and so on until 10 different concentrations are obtained (including the parent mixture in section 5.1).
   4. Dilute 20 μl of each concentration in THF with 180 μl of medium.
   5. Prepare a control solution with THF only; 20 μl of THF with 180 μl of medium in each measurement.
   6. Add 10 μl from the resulting solution (including the THF control), to each well that already contains 200 μl of the aforementioned solution of cells in the medium to give final concentrations of the compound of up to 200 mg/L (the concentration range can be changed according to the compound activity).
   7. You may observe some precipitation at the highest concentration applied depending on the compound solubility.
   8. Repeat each measurement of the compound 3x (3 lines in the plate of the same compound); each line contains: cells treated with the compound in 10 different concentrations, 1 well containing cells treated with solvent only for control and 1 well without cells for blank control.
   9. Incubate the loaded plate for 3 days at 37 °C in 5% CO₂ atmosphere.
6. Cytotoxicity measurement using the MTT assay
   1. Prepare the MTT solution by adding 1 g of MTT powder to a 200 ml solution of RPMI-1640 medium without phenol red. The stock solution can be divided to 15 ml tubes and stored in -20 °C.
   2. Add 20 μl of MTT solution to each well using 11-channel pipette (except to the blank control wells without the cells from step 4.10), and incubate the plate for additional 3 hr.
   3. Remove the medium solution from each well.
   4. Add 200 μl of isopropanol to each well to dissolve formazan. Stir the plate for 0.5 hr until homogeneousness is reached.
   5. Measure the absorbance at 550 nm for 200 μl of the aforementioned solution by a microplate reader spectrophotometer.
   6. Repeat each measurement (that includes 3 repetitions, see step 5.8) on 3 different days.
   7. Calculate the percentage of cell viability by deducting the blank control absorbance value from the measured value, dividing the result by the absorbance of the THF solvent control value and multiplying by 100%.
   8. Calculate IC₅₀ values using GraphPad Prism software (or equivalent).
   9. The reported IC₅₀ is the average of all IC₅₀ values collected on at least three different days, and the error value is the standard deviation.

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

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