Chapter II. Intracellular reaction in mitochondria for selective cancer therapy 19
2.3 Result and Discussion
2.3.1 Characterization of TPP-ALD, TPP-HYD and Hydrazone-2TPP
To confirm the intracellular reaction, we measured UV/Vis spectra and Fluorescence spectra of TPP- ALD (2), TPP-HYD (5), Hydrazone-2TPP.
As shown in figure 9, The TPP-ALD (2) and TPP-HYD (5) shows the absorbance in 275 nm, 259 nm respectively. And after hydrazone reaction, Hydrazone-2TPP was also check the UV/Vis spectra and Fluorescence. The result shows that the absorbance appear at the 364 nm and fluorescence maximum wavelength is 517 nm. In the fluorescence data, the fluorescence at 517 nm only showed high intensity about 5000-fold in Hydrazone-2TPP after reaction compare to TPP-ALD (2) and TPP-HYD (5). It means that reaction show the green fluorescence and it appear turn on process.
Figure 2.9.Characterization of molecular structure (A) UV/Vis spectra of TPP-ALD (2) of 500 μM in DMSO (λex 275 nm) (B) UV/Vis spectra of TPP-HYD (5) of 500 μM in DMSO (λex 259 nm) (C) Fluorescence spectra of TPP-ALD (2), TPP-HYD (5) and Hydrazone-2TPP with 500 μM in DMSO (λex 364 nm, λmax 517 nm).
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Wavelength(nm)
TPP_ALD
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Wavelength(nm)
TPP_HYD
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Wavelength(nm)
TPP_ALD_500uM TPP_HYD_500uM Hydrazone_2TPP_500uM
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High-performance equivalent chromatograph (HPLC) was used to verify that TPP-ALD (2) and TPP- HYD (5) are responding and generate Hydrazone-2TPP. In figure 10, the retention time between TPP- ALD (2) and TPP-HYD (5) was about 21 minutes, whereas the Hydrazone-2TPP was 25 minutes, and the retention time was later compared to the two substances. It means that after the reaction, the hydrophobicity is increasing. This will confirm that the response has progressed and the retention time has changed.
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Intensi ty
Retention time (min)
TPP-ALD TPP-HYD Hydrazone-2TPP
Figure 2.10. HPLC retention time for reaction confirm of TPP-ALD (2), TPP-HYD (5) and Hydrazone-2TPP in Acetonitrile.
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2.3.2 Cell viability of TPP-ALD, TPP-HYD and Hydrazone-2TPP
To check the cytotoxicity for cancer cell and normal cell, HeLa cell line and NIH-3T3 cell line were used. The hypothesis of this experiment was that each of the two non-toxic substances increased their toxicity through intracellular reactions, thus selectively targeting only cancer cells. However, as shown in figure 11, as a result of measuring cytotoxicity, TPP-HYD (5) showed low cytotoxicity as hypothesis, whereas TPP-ALD (2) showed an IC50 value of about 40 μM. It was confirmed that it was very toxic and judged that there was no selectivity both of cell line. Also, there were high toxicity in cancer cell line and normal cell in Hydrazone-2TPP. So, it has no selectivity and mixture of TPP-ALD and TPP- HYD also no selectivity. Furthermore, originally TPP-ALD has non-toxic, but it has also toxicity.
Figure 2.11. Cell viability analysis of NIH3T3 cell line and HeLa cell line (A) Treatment of TPP- ALD (2) with different concentration (0, 5, 10, 20. 40. 50 μM) for 24 h (B) Treatment of TPP-HYD (5) with different concentration (0, 5, 10, 20. 40. 50 μM) for 24 h (C) Treatment of hydrazone-2TPP with different concentration (0, 5, 10, 20. 40. 50 μM) for 24 h (D) Treatment mixture of TPP-ALD and TPP-HYD (2+5) with different concentration (0, 5, 10, 20. 40. 50 μM) for 24 h.
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2.3.3 Colocalization of TPP-ALD, TPP-HYD and Hydrazone-2TPP
The cells were labeled with Mitotracker Red, incubate with 100 μM TPP-HYD (5) for 2 h firstly and incubated with 100 μM TPP-ALD (2) for 3h. And Hydrazone-2TPP also treated with 100 μM for 3 h.
When the reaction occurred under laboratory conditions, Hydrazone-2TPP which is control, showed a wavelength range of 517 nm, and based on this, confocal laser microscopy was used to demonstrate the intracellular reaction in the mitochondria. Since both TPP-ALD (2) and TPP-HYD (5) have mitochondria targeting moiety (TPP), we expected these two substances to enter the mitochondria and react each other. Due to the high toxicity of TPP-ALD (2), 100 μM of low toxicity TPP-HYD was first incubated for 2 h. After that, the remaining TPP-HYD (5) was washed with DMEM, and then 100 μM of TPP-ALD (2) was incubated for 3 h. The remaining TPP-ALD was also washed and treated with Mitotracker red for 30 minutes to observe overlap. In figure 12, the green fluorescence was observed in the mixture of TPP-ALD (2) and TPP-HYD (5). And this green fluorescence overlaped very well with Mitotracker red, a red fluorescence, showing yellow. This tendency was also observed in Hydrazone- 2TPP (control). Hydrazone-2TPP is very toxic compare to others, so it was confirmed after 3 h incubation with 100 μM. This proved that intracellular reactions occurred in mitochondria. However, due to the great toxicity, we found that the morphology of mitochondria is being modified and dying.
And as seen in mitochondria red, it was confirmed that the staining was carried out inside the cell nucleus. As a result, it was confirmed that the two substances which are TPP-ALD (2) and TPP-HYD (5) put into each of the HeLa cells caused the intracellular reaction to fluorescence.
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Figure 2.12. (A) CLSM image to check the intracellular reaction in mitochondria treated with 100 μM TPP-ALD (2) for 2 h firstly and then treated with TPP-HYD (5) for 3 h. (B) treated with 100 μM Hydrazone-2TPP for 3h.
However, the cytotoxicity test confirmed that both Hydrazone-2TPP and TPP-ALD (2) were highly toxic and could not make a difference in toxicity between normal cells and cancer cells. Based on these conclusions, we redesigned the material which has non-toxic and optimized a substance that can give selective toxicity in normal cells and cancer cells, So we conducted experiments using Br-ALD (1), a previous step in TPP-ALD (2).
34 2.3.4 Optimize the intracellular reaction
2.3.4.1. Characterization of Hydrazone-TPP
Since TPP-HYD (5) was not originally toxic until 50 μM, TPP-HYD (5) was used as it is. However, TPP-ALD (2) was very toxic and needed optimization of TPP-ALD (2). Therefore, we used Br-ALD (1), a preliminary step in TPP-ALD (2) synthesis.
First, we check the UV/Vis spectra and Fluorescence spectra of Hydrazone-TPP which is mixture of Br-ALD (1) and TPP-HYD (5) because of the confirming the reaction. It showed absorption peak in UV/Vis spectra at 317 nm and fluorescence maximum intensity also appear in 414 nm wavelength. This wavelength is a little blue shift compared to That of Hydrazone-2TPP. TPP has influence the red shift of the fluorescence wavelength. Through this data, Hydrazone-TPP also react in experimental condition.
So we continued next experiment.
Figure 2.13. Characterization of molecular structure (A) UV/Vis spectra of mixture of TPP-ALD and TPP-HYD with 500 μM in phosphate buffer pH 8.1 (λex 317 nm) (B) Fluorescence spectra of TPP- ALD and TPP-HYD with 500 μM in phosphate buffer pH 8.1 (λmax 414 nm)
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Wavelength(nm)
Hydrazone_TPP
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Absorbance
Wavelength (nm)
Hydrazone_TPP
35 2.3.4.2 Cell viability of Hydrazone-TPP
After that, to determine the cytotoxicity, we checked the cell viability of Br-ALD (1), TPP-HYD (5) and mixture of Br-ALD and TPP-HYD (1+5). First, Br-ALD has treated using different concentration such as from 0, 10, 20 … to 100 μM for 24h incubation. But the cell viability was upper than 80 % except 80 μM and 100 μM because of very high concentration. Secondly, TPP-HYD also incubated for 24h using different concentration to check the cell viability in HeLa cell line. (Figure 14)
And finally, to confirm the intracellular reaction in mitochondria, we treated TPP-HYD for 1h incubation first. and to optimize IC50 value, TPP-HYD incubated with different concentration from 0, 5, 10 … to 80 μM. However, Br-ALD concentration was fixed with high concentration to 50 μM and 100 μM respectively (figure 15). Because the Br-ALD does not have mitochondria target moiety like TPP, we treated Br-ALD with high concentration which has non-toxic until 100 μM.
As a result, the figure 14 shows that according to increasing concentration of Br-ALD 50 μM to 100 μM, the cytotoxicity is also increased dramatically up to 5% in that 100 μM condition.
So, the data shows that the intracellular reaction occurred in the mitochondria because of first treated TPP-HYD, it can be entered to mitochondria first and after that Br-ALD entered to mitochondria and react each other. It occurred the cytotoxicity according to intracellular reaction.
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Concentration ()
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Cell viability (%)
Concentration (uM)
Figure 2.14. Cell viability in HeLa cell line (a) treated to Br-ALD at different concentration
(0,10,20...100 μM) for 24 h. (b) treated to TPP-HYD at different concentration (0,10,20…100 μM) for 24 h.
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Figure 2.15. Cell viability in HeLa cell line. (A) TPP-HYD treated with different concentration (0, 5, 10...80 μM) for 1 h firstly, and washed with DMEM and then Br-ALD treated with 50 μM for 24 h. (B) TPP-HYD treated with different concentration (0, 5, 10...80 μM) for 1 h firstly, and washed with DMEM and then Br-ALD treated with 100 μM for 24 h.
To check the selective cytotoxicity, the IMR90 cell line which is normal cell line was used. In this case, like HeLa cell line, firstly cytotoxicity of Br-ALD and TPP-HYD were checked. In the Figure 16, it shows that both of them also had non-toxicity in the IMR90 cell until 80 μM concentration. And to confirm the cytotoxicity through intracellular reaction, treat Br-ALD and TPP-HYD with same method like HeLa cell line. (Figure 17)
Figure 17 also shows cytotoxicity of Hydrazone-TPP, a mixture of Br-ALD and TPP-HYD. Unlike HeLa cells, we found that cytotoxicity does not decrease significantly with the concentration of TPP- HYD in normal cells (IMR90 cells). And even at high concentrations of 80 μM, we can see that the cell viability is little toxic. In the next figure, we compared the cell toxicity of normal cells and the HeLa cells, and compared it with the other normal cells, HEK293T cells.
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Figure 2.16. Cell viability in IMR 90 cell line (a) Br-ALD treated with different concentration (0, 5, 10...80 μM) for 24 h. (b) TPP-HYD treated with different concentration (0, 5, 10...80 μM) for 24 h.
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Cell viability (%)
Concentration ()
Figure 2.17. Cell viability in IMR 90 cell line. TPP-HYD (5) treated with different concentration (0, 5, 10...80 μM) for 1 h firstly, and washed with DMEM and then Br-ALD (1) treated with 50 μM for 24 h.
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Figure 18a is a graph comparing the cytotoxicity of the HeLa cell line, a cancer cell, and the IMR90 cell line, a normal cell. In this graph you can see a significant difference in toxicity from 5 μM. In HeLa cells, the viability dropped sharply to 60% from 5μM, whereas in IMR90 cells, the toxicity increased as the concentration increased. We believe that this can target cancer cells by selectively increasing toxicity only to cancer cells. In addition, we conducted a toxicology test using another normal cell, MEK293T cell line, to determine if the toxicity was weak only on IMR90 cells. (figure 18b) As a result, cell viability such as IMR90 can be found in the HEK293T cell line up to 30 μM. However, since cytotoxicity increases from 50 μM, it can be considered that the concentration is high and the toxicity increases. However, the HEK293T cell line is also more toxic to cancer cells than the HeLa cell.
As the results, the viability of IMR90 cell line is well marked higher compared to HeLa cell line. The IC50 value of IMR90 is about 60 μM and HeLa cell is about 15 μM. It means that cancer cell line can be selectively toxicity.
(a) (b)
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Concentration (uM)
IMR90 HeLa
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Figure 2.18. Cell viability between normal cell line and cancer cell line. (A) Comparison with IMR90 cell line and HeLa cell line. TPP-HYD (5) treated with different concentration (0, 5, 10...80 μM) for 1 h firstly, and washed with DMEM and then Br-ALD (1) treated with 50 μM for 24 h. (B) comparison with HEK293T cell line and HeLa cell line. TPP-HYD (5) treated with different concentration (0, 5, 10...80 μM) for 1 h firstly, and washed with DMEM and then Br-ALD (1) treated with 50 μM for 24 h.
39 2.3.4.3 optical properties of intracellular reactions.
We subsequently studied mitochondrion specificity of Hydrazone-TPP in HeLa cells by colocalization with Mitotracker red depending on time. We treated Br-ALD and TPP-HYD according to cytotoxicity data. TPP-ALD was incubated for 1 h using 40 μM frist, and then Br-ALD was treated on 50 μM according to time increasing like 30 min, 1 h and 3 h. and then treated Mitotracker red for 30 min lastly.
And then the fluorescence was checked with FV1000 laser confocal scanning microscope.
The fluorescence of Hydrazone-TPP, which is mixture of Br-ALD and TPP-HYD, is not appeared 30 min. however, according to increasing time, the fluorescence intensity is gradually increased. But total intensity of fluorescence is weak compare to Hydrazone-2TPP. (Figure 19) In 3 h data, the red signal from mitotracker red is well overlapped with green fluorescence from Hydrazone-TPP, with a Pearson correlation coefficient of 0.72.
Figure 2.19. Confocal image for intracellular reaction in mitochondria. TPP-HYD (5) treated with 40 μM for 1 h and then Br-ALD (1) treated with 50 μM for different incubation time. (A) 30 min (B) 1 h (C) 3 h in HeLa cell line and the coefficient is 0.72
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2.3.6 Mitochondrial dysfunction of intracellular reactions.
To prove mitochondrial dysfunctions, we use TMRM to estimate mitochondrial membrane potential differentiation by Hydrazone-TPP and MitoSox to estimate the ROS generation by Hydrazone-TPP.
Because the mitochondria membrane was damaged by Hydrazone-TPP, the red signal from TMRM was diminished after treated Hydrazone-TPP. (Figure 20) As opposed to TMRM, the red signal from MitoSox was occurred in the nuclei after treated Hydrazone-TPP. as a result, after treating the Hydrazone-TPP, the mitochondria membrane is destroyed and the ROS is released, indication that the mitochondria are dysfunction. (Figure 21)
Figure 2.20. CLSM image showing mitochondrial membrane depolarization analysis by using TMRM in HeLa cells with 40 μM TPP-HYD (5) treatment for 1h and 50 μM Br-ALD (1) treatment for 3h.
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Figure 2.21. CLSM image to check the mitochondria-ROS production using Mito-Sox in HeLa cells with 40 μM TPP-HYD (5) treatment for 1h and 50 μM Br-ALD (1) treatment for 3h.
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2.3.7. Apoptosis experiment
And finally, to identify apoptosis, we had the FACS experiment.
All sample processing methods in the FACS data were performed in the same manner as the cytotoxicity test. In the cytotoxicity test, TPP-HYD was first treated for 1 hour and Br-ALD was treated for 24 hours.
Therefore, four experiments were conducted in FACS experiment. First, apoptosis was confirmed when TPP-HYD was incubated for 1 hour, apoptosis was confirmed when Br-ALD was incubated for 24 hours, and Hydrazone-TPP was formed through intracellular reaction. As a result, when only 20 μM of TPP-HYD was treated, necrosis and early apoptosis rarely occurred and the probability of late apoptosis was 5.57%, indicating that almost no apoptosis occurred. Second, in Figure 13c, apoptosis was confirmed after 24 hours of incubation of Br-ALD 50 μM. Similar to the data in TPP-HYD, necrosis and early apoptosis do not occur, and only the percentage of late apoptosis can be seen to increase to 18 percent. I think this is due to the high concentration of Br-ALD. Figures c and d show apotposis after formation of Hydrazone-TPP through concentration-dependent intracellular reactions. As a result, the necrosis and early apoptosis increased only slightly, and late apoptosis increased to 43.79% when TPP- HYD was treated with 20 μM. This confirmed that intracellular reactions occurred, causing early apoptosis. In addition, increasing the concentration of TPP-HYD to 40 μM showed that the percentage of late apoptosis increased to about 60 percent. As a result, it was confirmed that the intracellular reaction in HeLa cells produced hydrazone-TPP, which induces late apoptosis. (Figure 22)
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Figure 2.22. Flow cytogram image representing the apoptosis assay based on annexin V-FITC and PI staining of HeLa cells treated with (A) control (B) 20 μM TPP-HYD (5) for 1h (C) 50 μM Br-ALD (1) for 24h (D) 20 μM TPP-HYD (5) for 1h and then treated 50 μM Br-ALD (1) for 24h (E) 40 μM TPP- HYD (5) for 1h and then treated 50 μM Br-ALD (1) for 24h
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