Synthesis, cytotoxicity, and long-term single dose anti-cancer pharmacological evaluation of dimethyltin(IV) complex of N(4)-methylthiosemicarbazone (having ONS donor ligand)

Figures & data

Figure 1. (A) Synthesis of dimethyl tin(IV): 5-bromo-2-hydroxy benzaldehyde-N(4)-methyl thiosemicarbazone; (B) IR spectrum; (C) UV-visible spectrum of dimethyltin(IV)-N(4)-methylthiosemicarbazone complex; (D) ¹H NMR spectrum; (E) ¹³C NMR spectrum.

Figure 2. The compound screening procedure: first, synthesized compound was tested in three cancer cell lines, MCF7, HCT116, A549, and normal cell lines EAhy926, at multiple concentrations of 6.25 to 200 μg/ml.

Note: The percentage viability of cells was measured as short-term effects and changes in clonogenic potential and 3D tumor spheroid assay as long-term effects of the synthesized compound. Two criteria were used to select the concentration of compound for further analysis: IC₅₀ of the compound that reduce cell viability to 50% of control and high–low quantity of IC₅₀. Next, the compound was used as double, half, and quarter IC₅₀ concentrations to investigate short-term effects (migration, nuclear condensation, mitochondrial potential) for the ability to induce apoptosis and long-term effects (clonogenic, 3D tumor) to influence anticlonogenic, antiaggregation (3D tumor model) activity.

Figure 3. Photomicrographic images of different cancer cell lines and normal endothelial cells treated with BHBM tamoxifen, and 5-fluoruracil (5FU).

Note: Images were obtained from EVOS inverted microscope after 48 h of treatment. MCF7 (A: untreated, B: treated), HCT116 (C: untreated, D: treated), A549 (E: untreated, F: treated) cells were incubated with BHBM for 48 h at different doses. Percent inhibition of cell proliferation (G) was obtained and values were expressed as μg/ml. MCF7 (X), HCT116 (Y), A549 (Z) cells were incubated with tamoxifen and 5-FU for 48 h at different doses. Percent inhibition of cell proliferation (J) was obtained and values were expressed as μg/ml. Results are presented as mean ± SD of three separate experiments (n = 6).

Table 1. IC₅₀ and selectivity index (SI) of the various melanoma (cancer) cells

Samples	IC50 (μg/ml) on MCF7	IC50 (μg/ml) on HCT 116	IC50 (μg/ml) on A549	IC50 (μg/ml) on EAhy926	IC50 (μg/ml) on MDCK	Selectivity Index (SI)			NO generation effect in HCT116 (IC50 μg/ml)
						MCF7	HCT 116	A549
BHBM	6.0±0.57	4.0±0.65	20±0.54	40±1.2	55±1.7	7±2.5	10±2.8	2±0.8	4.5±1.7
Tamoxifen	7.0±0.98	–	–	20±2.5	15±2.87	3±1.5	–	–	ND
5 fluorouracil	–	2.0±0.75	3.0±0.78	20±2.9	10±2.5	–	10±1.7	6±0.1.9	ND

Note: SI is the ration of IC₅₀ for normal cell line and cancerous cell line after 48 h of treatment. IC₅₀ was measured after 48 h treatment with compounds using MTT staining and it was expressed as % of vehicle (0.05% DMSO)-treated control. For comparison normal cells endothelial cell line EAhy926 and normal epithelial cell line MDCK and effects of standard chemo drugs Tamoxifen and 5-fluorouracil were used. Results are presented as mean ± SD of three separate experiments (n = 6).

Figure 4. Antimetastatic functional assay: migration.

Note: Migration Effect of BHBM on proliferation and migration of MCF7 (I). Effect of BHBM on MCF7 proliferation. BHBM inhibited MCF7 proliferation in dose-dependent manner with IC₅₀ 6 μg/ml (n = 6, values are in mean ± SD). Due to the successful migration of MCF7 in untreated group, the wound is almost closed after 18 h, whereas in BHBM-treated group, the wound remained open even aft4er 24 h incubation. BHBM (3 μg/ml) caused significant inhibition of MCF7 migration. At a concentration of 6 μg/ml, BHBM caused dislodgement of monolayer of MCF7 (indicated by the arrows) with almost complete inhibition of migration. BHBM also successfully arrested the colon cancer HCT116 cells (II) and lung cancer cell line A549 (III) proliferation and migration for more than 36 h with an administration of a single dose. Antimigratory effects of BHBM were found as dose dependently. This effect showed a linear correlation between cytotoxicity and percentage wound closure. Values were expressed as means ± SD.

Figure 5. BHBM at 1-12 μg/ml negatively influenced the clonogenic growth of melanoma cells.

Note: MCF7 cells were treated with BHBM 12 μg/ml (I-A), 6 μg/ml (I-B), 3 μg/ml (I-C), and 1.5 μg/ml (I-F), DMEM media with DMSO as a Negative Control (I-E), and 5 μg/mL of tamoxifen as a Positive Control (I-D). Cell colonies were stained with crystal violet and counted. Anticlonogenic activity of BHBM was expressed as percentage of control treated with vehicle (0.05% DMSO). Experiments were performed in triplicate. HCT116 cells were treated with BHBM 8 μg/ml (II-A), 4 μg/ml (II-B), 2 μg/ml (II-C), and 1 μg/ml (II-F), DMEM media with DMSO as a negative control (II-E), and 5 μg/mL of 5-FU as a positive control (II-D). Cell colonies were stained with crystal violet and counted. Anticlonogenic activity of BHBM was expressed as percentage of control treated with vehicle (0.05% DMSO). Experiments were performed in triplicate.

Figure 6. Three-dimensional (3D) tumor spheroid-based functional assay: invasion. 48-well agar-coated flat-bottomed plates were used to generate A549 spheroids (a single spheroid per well).

Note: Images were captured at four days intervals (data not shown) using an EVOS microscope. Analysis was carried out using Image-J software and percent inhibition tumor aggregation was obtained in 12 days. The comparative effects of BHBM on in vitro A549 (lung) tumor in hanging drop assay. Values were measured as means ± SD. Results are presented as mean ± SD of three separate experiments (n = 6).

Figure 7. Photomicrographic images of the MCF7 in nuclear condensation assay (I). Staining of cultures by the use of Hoechst revealed the damaged DNA as an indicator of apoptosis. Higher cell death was found at 24 h (data not shown) than the tamoxifen. BHBM showed almost similar apoptotic index with mid and high doses at 12 h. The medium dose 6 μg/ml was more effective than the higher dose. BHBM at very high dose was not pronounced as anticancer activity. Values were determined as means ± SD. Photomicrographic images of the MCF7 in mitochondrial membrane potential (II).

Note: The rhodamine staining of nuclei reflects the intracellular generation of the cancer cells after treated with BHBM. Photomicrographic images of the cells showed the higher efficiency of the BHBM (higher intensity of rhodamine uptake cells) to disrupt the cancer cells at the dose of 6 μg/ml during 6 h treatment than 12 h. Percent apoptotic index was measured from the number of apoptotic cells (shiny cells) to the total number of cells in each selected microfield. Values are measured as means ± SD. Results are presented as mean ± SD of three separate experiments (n = 6).

Figure 8. Visualization of Ligand and protein interaction profile: surface visualization (I-A) and cartoon display (I-B) of NF-κB and active site residue interaction of protein NF-κB (I-D, I-F, I-H).

Note: Molecular docking was performed in Autodock Vina. The binding energy obtained from docking analysis reflected the anticancer activity that correlates QSAR model. Descriptor Solvent accessible surface area (SASA), surface area (SA) of BHBM was generated by CODESSA software (X, Y, Z). SASA is very important to correlate the molecule structure with the anticancer activity. QSAR model was developed in CODESSA using this SASA descriptor by the multiple linear regression (MLR) method. Calculated efficiency versus the experimental efficiency of the training set molecules were plotted by MLR method (H). Results are presented as mean ± SD of three separate docking experiments.

Figure 9. Anticancer activity of BHBM through the induction of apoptotic cell death.

Notes: In addition, the illustrated mechanism of action depicts that Dimethyl tin(IV) complex BHBM could produce multiple site of action to stop the nutrient supply to the cancer cells and weaken the cancer cells with the specific interaction to the active amino acids. BHBM is not highly toxic to the cells, but the cell death occurs due to its multiple hit of action. Chelation, cytotoxic O2 radicals, methylation of glutamine, preventing signals to synthesize the DNA, resonance impact of halide group, hydrogen bonding capacity and so on.

Scheme 1. Synthesis of dimethyltin(IV)-5-bromo-2-hydroxybenzaldehyde-N(4)-methylthiosemicarbazide complex.

Table 2. pKa and LogD physicochemical properties of BHBM

Compounds	pKa	LogD	Antioxidant			Caspase
			DPPH EC50 (μg/mL)	FRAP (nmol Fe+2 eq./mg compound)	ROS	Caspase 3	Caspase 8	Caspase 9
BHBM	7.38±0.12	−0.92±1.5	320.50±12.18	54.83±0.48	66.23% decrease	0.735±0.87	0.71±0.58	0.661±0.97
Ascorbic acid	–	–	7.30 ±0.01	–	–	–	–	–
control	–	–	–	–	–	0.588±0.43	0.651±1.2	0.551±0.35

Note: Effect of BHBM on levels of caspases 3/7, 8, and 9. A significant increase in the apoptotic markers, caspases 3/7, 8 and 9 was observed after 48 h to HCT116 cells. BHBM at 4 and 8 μg/mL significantly increased the levels of caspases 8 and 9. Higher concentration of BHBM had an effect on caspase 8 and 9 induction that was comparable to the control. The results are mean values ± SEM (n=3). Free radical scavenging activity of the BHBM measured by DPPH assay. Results are expressed as mean ± SEM (n=3). Effect of the BHBM on ROS level. The relative ROS fluorescence intensity in cultures of HCT116 cells treated with 4 and 8 μg/mL of BHBM over 48 h was measured using the ROS assay reagent. HCT116 cells incubated with two different concentrations of BHBM showed a dose-dependent decrease ROS levels compared to the control. Results are means ± SEM of three experiments (n = 6).

Table 3. QSAR model generated by CODESSA for predicted pIC₅₀ and calculated IC₅₀

Method	Property	No. of Descriptor	R^2	CVR^2	F	Structure Set	Descriptor Set	s^2	SEE	p (F)	CVSE
MLR	Efficiency	20	0.711	–3932.84	0.11	22 training set	Solvent accessible surface area	2.75	0.35	0.99	40.45

Note: Statistical data shown the correlation coefficient R² value 0.711 indicates structure activity relationship with the training set molecules were strongly agree to quantify the anticancer activity of the BHBM. Standard error of the estimate (SEE), variance ratio (F), p(F), s², CVR², CVSE also were shown in the table.