Effect of advanced oxidation and amino acid addition on antioxidant capability, iron chelating property and anti-cancer activity of tannic acid

2.1 Materials

Ferric chloride hexahydrate (FeCl₃·6H₂O) was purchased from Fisher Chemicals. Tannic acid (C₇₆H₅₂O₄₆) was purchased from Loba Chemie. Roswell Park Memorial Institute (RPMI) 1640, Dulbecco’s modified Eagle’s medium Ham’s F-12 (DMEM/F-12), Penicillin-streptomycin and Trypsin-EDTA were purchased from Caisson Laboratories. Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific. Collagen type I, ribonuclease A (Rnase A), triton X-100, and propidium iodide (PI) were purchased from US Biological. Dimethyl sulfoxide (DMSO), potassium bromide (KBr), and 7-dichloro- fluorescein diacetate, (H2DCFDA) were purchased from Sigma-Aldrich. L-Lysine, (C₆H₁₄N₂O₂), L-Phenylalanine (C₉H₁₁NO₂), 3-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) were purchased from Bio Basic Inc. Ethyl alcohol and potassium dihydrogen phosphate (KH₂PO₄) were purchased from QRec. Di-Sodium hydrogen phosphate dihydrate (Na₂HPO₄) and tri-sodium citrate were purchased from RCI Labscan Limited. Potassium chloride (KCl), Sodium choride (NaCl), L-cysteine (C₃H₇NO₂S), and DMSO‑d₆ was purchased from Merck. Lymphoprep^TM was purchased from Axis-Shield.

2.2 Oxidation and amino acid addition of TA

In order to show that chemical structure of tannic acid can be easily changed in a physiological-like environment, TA was allowed to incubate in PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) with and without amino acids such as lysine, phenylalanine, and cysteine. Typically, in order to allow oxidation reaction of TA, TA (0.01 mmol) was incubated in 30 mL of the PBS at 37 °C for different lengths of time. In case of amino acid modified TA, TA (0.01 mmol) was incubated in PBS containnig amino acids (0.03 mmol) at 37 °C for different lengths of time. The obtained solutions were subjected to fllter thru 0.2 µm sterile filter and were further kept at 4 °C for cellular experiments. In addition, the solutions of TA containing amino acids were subjected to purification by Slide-A-Lyzer dialysis Cassettes (2 K MWCO) and were further freeze-dried for further characterizations by FITR and NMR.

2.3 Characterization of different TA samples

UV–vis spectra were determined with Agilent UV–vis spectrophotometer (Agilent 8453, China). The functional groups of different TA samples were investigated by a Fourier transform infrared (FTIR) spectrophotometer (Bruker Tensor 27, Germany) with KBr as the diluting agent over the wavenumber 4000–400 cm⁻¹. Modifications of chemical structure of TA by amino acids were characterized by ¹H NMR spectroscopy (400 MHz, Bruker AVANCE TM, Germany) with DMSO‑d₆ as the solvent. Mophology of iron-tannic complexes was determined by transmission electron microscope (TEM, JEM-2010, JEOL, Japan).

2.4 Interaction of different TA samples with NTBI in different media

At first, ferric-citrate complexes were prepared in order to use as NTBI model. In typical, 10 mmol of tri-sodium citrate was mixed with 5 mmol of ferric chloride under vigorous stirring. The resulting pale-yellow solution was allowed to equilibrate for 30 min and further subjected to sterilization by using syringe filter (0.2 μm in diameter). To investigate iron binding efficiency of different TA samples, certain amounts of different TA samples (10 μM in final concentration) were mixed with different concentrations of ferric-citrate complexes in different media, including PBS buffer and PBS containing 10% FBS. The UV–Vis absorption spectra were recorded on a UV–vis spectrophotometer (Agilent 8453) at different lengths of incubation time. The iron binding capability of TA samples was obtained by measuring the absorbance of ligand-to-metal charge transfer band at ∼525 nm.

2.5 DPPH assay

The antioxidant properties of different TA samples, different amino acids, and ascorbic acid were analyzed by using DPPH assay. In typical experiment, PBS solutions containing different samples (10 μM in final concentration) were mixed with DPPH reagent, and were further incubated for 30 min in the dark. After that, the absorbance of the samples was measured at 520 nm using PBS as blank. The radical scavenging activity was calculated according to the following equation as; $\mathit{DPPH}scavenging\left(\%\right)=[\left(A_{\mathit{control}}-A_{\mathit{sample}}\right)/A_{\mathit{control}}]\times 100$ , where $A_{\mathit{control}}$ and $A_{\mathit{sample}}$ are the absorbance of blank DPPH solution and sample solution, respectively.

2.6 Protein precipitation assay

In order to examime protein precipitation induced by different TA samples, different concentration of TA samples were incubated with FBS solution for a week (7 days) at 37 °C under 5% CO₂ atmosphere. The protein precipitates were directly observed by naked eye.

2.7 Cell culture

Doxorubicin-resistant human leukemic cells (K562/ADR) were maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS and 1% penicillin-streptomycin solution and maintained at 37 °C under 5% CO₂ atmosphere.

Alpha mouse liver 12 (AML-12) cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% FBS and 1% penicillin–streptomycin solution and were maintained in a 5% CO₂ atmosphere at 37 °C.

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human peripheral blood by density gradient centrifugation using Lymphoprep^TM according to manufacturer’s instructions. The isolated cells were maintained in RPMI 1640 medium with 10% Fetal bovine serum, 1% Penicillin-streptomycin solution and maintained at 37 °C under 5% CO₂ atmosphere. Ethical approval for PBMC experiments was obtained from the AMS Human Research Ethics Committee (AMSEC-61EX-046).

2.8 Cell counting assay

K562/ADR and PBMC cells were seeded in 24-well plates (5 × 10⁴ cells/well) at 37 °C under 5% CO₂ atmosphere overnight. Next, the cells were treated with different TA samples for different lengths of time. At the desired time, the cells were counted by using a flow cytometer (Beckman Coulter's Epics XL-MCL). For the analysis, gated population (only lymphocytes for PBMC) was counted and the cell viability was relatively calculated as the percentage of untreated cells (control)

2.9 MTT assay

AML-12 cells were seeded in 24-well plates (5 × 10⁴ cells/well) and were incubated overnight. Then, the cells were treated with different TA samples for 48 h and 72 h. After treatment, the cells were washed with PBS and incubated with 5 mg/ml of MTT solution for 4 h. Next, the cells were washed with PBS and the intracellular formazan was dissolved in DMSO. The absorbance of the formazan solution was measured with a microplate-reader (Biotek Synergy H4) at 570 nm using DMSO as the blank. The cell viability was relatively calculated as the percentage of untreated cells (control).

2.10 Cell cycle analysis and apoptosis determination

The cells were seeded in 24-well plates (5x10⁴ cells/well) and were incubated overnight. The cells were treated with different TA samples for different lengths of time (10 μM for TA, TA + Lys and TA + Cys, 20 μM for Taox, and TA + Phe). After the treatment, the cells were harvested and washed twice with PBS solution. Next, the cells were fixed with 70% ethanol for 2 h at 4 °C. The fixed cells were incubated with 5 μL of 10% Triton X, 50 μL of 2 mg/mL RNase A, and 5 μL of 1 mg/mL PI for 20 min at 37 °C. The DNA content of the cells in different cell cycle phases and fractional DNA (apoptosis) was measured with the flow cytometer (PI channel) and the cell cycle distribution was analyzed by using Flowing Software 2.5.0. The percentage of cells in each cell cycle phase was quantified by using markers set within the analysis program.

2.11 Flow cytometry measurment of intracellular ROS (DCFDA staining assay)

The cells were seeded in 24-well plates (5x10⁴ cells/well) and were incubated overnight. The cells were treated with different TA samples for different lenghts of time (10 μM for TA, TA + Lys and TA + Cys, 20 μM for TAox and TA + Phe). After the treatment, the cells were washed twice with PBS and were then subjected to intracellular ROS measurement by incubating with 0.1 μM H2DCFDA at 37 °C in an incubator for 30 min. Afterward, green fluorescence intensity of DCF (FL1) of the stained cells was measured with the flow cytometer.

2.12 Flow cytometry measurment of lysosome (Acridine orange staining assay)

The cells were seeded in 24-well plates (5x10⁴ cells/well) and were incubated overnight. The cells were treated with a toxic dose of different TA samples for different lengths of time (10 μM for TA, TA + Lys and TA + Cys, 20 μM for TAox and TA + Phe). After the treatment, the cells were washed twice with PBS and were then subjected to lysosome measurment by incubating with 1.25 μM acridine orange at 37 °C for 10 min. Next, red fluorescence intensity of AO (FL3) of the stained cells was measured with the flow cytometer.

3.1 Oxidation and amino acid addition of TA

It has been reported that catechol in polyphenols can be oxidized to quinone groups in the presence of oxygen (Dai et al., 2019; Xiang et al., 2017). Herein, we attempted to investigate oxidation reaction of tannic acid dissolved in a simple physiological buffer (PBS buffer, pH7.4). Initially, we measured UV–Vis spectra of TA dissolved in PBS for different lengths of incubation time and compared that to those in DI water. Considering the spectrum of TA in DI water (Fig. 1a), two absorption peaks are clearly observed at 210 nm and 275 nm. Obviously, when we incubated TA in PBS for 10 min, absorption peaks of at 275 nm has a red shift toward 325 nm, which is attributed to deprotonated catechol of TA. As the time passed, significant change in spectra shape of TA in PBS is observed, especially the presence of the absorption peak at 235 nm, which is attributable to oxidized tannic acid. From these results, we can conclude that TA can be oxidized in physiological conditions into semiquinone and o-benzoquinone. Possible oxidation reaction of TA is shown in Fig. 1(inset). It should be noticed that the oxidation yield cannot completely occur in that condition. At least 3 species are present, including (i) protonated TA, (ii) deprotonated TA and (iii) oxidized TA, depending on TA concentration and time of incubation (Rahim et al., 2014). Figure S1 shows UV–Vis spectra of different concentrations of TA dissolved in DI water and PBS for different lengths of incubation time. Clearly, there is no change in spectra shape of TA from dissolving in DI water, indicating no oxidation reaction occurred. However, dramatical change in spectra shape is observed as dissolved in PBS, indicating that oxidation easily occurred in that physiological condition.

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Fig. 1

(a) UV–Vis spectra of TA (1 μM) incubated in DI water and PBS for different lengths of time (inset; possible mechanisms for TA oxidation), (b) photographic image of different TA (0.33 mM) incubated in DI water (TA), PBS (TAox), PBS containing lysine (TA + Lys), PBS containing phenylalanine (TA + Phe), PBS containing cysteine (TA + Cys) (inset; possible mechanisms for amino acid addition reaction of TA), (c) UV–Vis spectra of TA (0.33 mM) incubated in PBS (Taox), PBS containing lysine (TA + Lys), PBS containing phenylalanine (TA + Phe), PBS containing cysteine (TA + Cys), (d) photographic image of different TA samples (0.2 mM and 2 mM) incubated in 10% FBS for a week along with their precipitates (obtained by using 2 mM TA samples).

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It is known that the oxidized catechol can react with various nucleophiles (e.g. -SH, -NH₂) in biomolecules via Michael addition reaction (Liu et al., 2019). Therefore, we aimed to know whether such reaction occurred during autoxidation of TA in PBS. We incubated TA in PBS pH 7.4 containing amino acid (L-lysine, L-cysteine, and L-phenylalanine) at 37 °C for 5 days. It is clearly seen that there are changes in color of solution of TA in PBS (TAox), TA in PBS containing lysine (TA + lys), TA in PBS containing phenylalanine (TA + phe,) and TA in PBS containing cysteine (TA + cys), compared to those in DI water (Fig. 1b). Possible reactions between an oxidized TA and amino acid are shown in Fig. 1b (inset). In order to confirm the existence of amino acid conjugated TA, we purified the conjugated TA samples via a dialysis membrane, and subjected it to further chemical analysis. According to spectroscopic analysis, there is some change in spectra shape of oxidized catechol (230–260 nm) in TA containning amino acid, compared to TA without amino acid, implying that electronic structure of oxidized TA is disturbed by interaction of amino acid (Fig. 1c). In addition, it should be noticed that TAox, TA + lsy, TA + phe, TA + cys seem to have absorption band in visible light region (400–550 nm), which are in accordance with their deep color. According to FTIR and ¹H NMR measurements, all conjugated TA shows the characteristic vibration peaks of amino acid (e.g. N-H stretching, C-N stretching and COO– bending), and several proton signals of amino acid(Gauri et al., 2013) (Figure S2-S6). Above results confirm the formation of TA conjugates in physiology buffer containning amino acid.

3.2 Protein precipitation

It has been reported that TA is capable of inducing protein precipitation via the same mechanism mentioned above. Therefore, we performed protein precipitation assay by incubating different TA samples in fetal bovine serum (FBS) for 7 days. As seen in Fig. 1d, no precipitation is observed when using 0.2 mM in all TA samples, while those of higher concentration (2 mM) are found to induce protein precipitation in FBS, except TA + Cys. This suggests that modification of TA structure with cysteine is capable of reducing non-specific binding between protein in the serum and TA. This is in agreement with other reports that cysteine is considered to be a zwitterionic amino acid, which is able to reduce non-specific interaction (Choi et al., 2007). From this result, we might take an advantage of the reduction of protein precipitation by pre-treatment with TA along with cysteine to develop TA conjugates for various applications including nutraceuticals, functional foods, and so on.

3.3 Iron-chelating property

Previously, we demonstrated that TA was able to bind to small iron complexes in physiological media, which can be utilized as a natural iron chelator for chelation therapy (Phiwchai et al., 2018). Herrin, we investigated iron binding capacity of different TA samples in PBS and 10% FBS by measuring absorption peaks of ligand-to-metal charge transfer (LMCT) bands of iron-tannic complexes. Typical UV–Vis spectra exhibits the LMCT peak of iron-tannic complexes at ∼500–550 nm, corresponding to a high stable tris-structure of iron-tannic complexes (Fig. 2a,b). The corrected absorbance values are found to increase as the concentration of ferric-citrate complexes increases in both PBS and FBS, demonstrating their capability for iron chelation. By measuring the absorbance of LMCT of different TA samples containing different concentrations of ferric-citrate complexes, we found a good linear correlation between the absorbance and concentration of ferric-citrate complexes over the range of 0–100 μM in all TA samples (Fig. 2a and b), implying that alterations of chemical structure of TA by oxidation and amino acid addition do not strongly affect the ability for iron chelation. This result suggests that all TA samples can be applicable as efficient iron chelators for iron chelation therapy as well as sensors for NTBI detection. Unlike other clinical iron chelators such as deferoxamine, deferiprone and deferasirox, TA samples are able to bind a higher content of iron per molecule and have capability of generating nanosized iron tannic complexes via self-assembly processes (Phiwchai et al., 2018). Fig. 2c shows typical TEM images of nanosized iron-tannic complexes obtained by mixing NTBI with different TA samples. The nanosized complexes provide additional benefits for prevention and treatment such as induction of autophagy and related processes (Phiwchai et al., 2019), as well as the promotion of autophagic cell death (Pilapong et al., 2020).

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Fig. 2

Typical UV–Vis spectra of TA containing different concentration of ferric citrate complexes, and plots of corrected absorbance (Abs_525nm-Abs_900nm) versus concentration of ferric citrate complexes measured from different TA samples incubated in (a) PBS and (b) 10 %FBS, (c) typical TEM images of nanosized iron-tannic complexes obtained by mixing iron with different TA samples using 3:1 mol ratios of Fe:TA.

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3.4 Antioxidant capability

Because TA is considered as an anti-antioxidant, we comparatively determined radical scavenging activity of different TA samples, pure amino acids, and ascorbic acid, and reported as a percentage of DPPH scavenging (Lou et al., 2018). The results show that the percentages of DPPH scavenging of TA and TAox are much higher than those observed in other samples for example TA containing amino acids, pure amino acids, and ascorbic acid, indicating a potent antioxidant activity of TA and oxidized TA (Fig. 3a). As is known, assessment of antioxidant activity by DPPH assay rely on capability of the sample to scavenge the DPPH radicals by donating electrons or hydrogen atoms (Formagio et al., 2014). The strongest activity found in oxidized TA is associated with better electron donating capability of the oxidized form of TA. However, TA undergoing amino acid addition reaction is found to decrease antioxidant activity, implying that chemical structure modification of TA by amino acid alters electron donating capability of TA. Surprisingly, however, complex compounds between NTBI and different tannic samples exhibit stronger scavenging activity than TA samples alone (Fig. 3a), which are in good agreement with several tannic nanoplatforms from previous reports (Sahiner et al., 2016; Wei et al., 2020). Stronger scavenging activity found in nanosized iron-tannic complexes may be due to the presence of iron in the tannic complexes that strengthen the electron withdrawing capacity of TA (Yang et al., 2017).

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Fig. 3

(a) Percentages of DPPH scavenging activity of ascorbic acid, different amino acids, different TA samples (b) Percentages of DPPH scavenging activity of different TA samples and TA samples containing NTBI.

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3.5 Anti-cancer activity

Similar to other polyphenols, TA has also been shown to exhibit anti-cancer activity (Kim et al., 2019; Nie et al., 2016; Sp et al., 2020). Herein, we aimed to investigate anti-cancer activity of un-modifed TA and TA that underwent advanced oxidation and amino acid addition. At first, we investigated the effect of different TA samples on cancer cell proliferation. It is seen in Figure S7 that proliferation rate of K562/ADR cells is different in each samples, depending on the concentration. From these results, IC50 values were determined according to dose–response curves, and the results are shown in Table 1. TAox and TA + Phe are found to exhibit lower anti-cancer activity whereas those for TA + Lys and TA + Cys are better, as compared to un-modified TA, depending on time of incubation. This indicates that advanced modifications of chemical structure of TA play a critical role on anticancer activity. It is important to notice that amino acid used in this experiment have no significant effect on cell proliferation. Therefore, it would be solely implied that any change in cell proliferation is caused by modification of chemical structure of TA during their autooxidation and amino acid addition. The results mentioned above demonstrate that alteration of chemical structure of TA strongly affect anticancer activity of TA. However, potential degradation of TA samples mediated by chemicals or enzymes need to be taken into account because it might alter their toxicity in both in vitro and in vivo (Manach et al., 2004; Nakamura et al., 2003). As far as toxicity is concerned, cytotoxicity of different TA samples against normal cells was also carried out. In our study, cytotoxicity against normal cells was conducted by using peripheral blood mononuclear cells (PBMCs) and normal hepatocytes (AML-12) as in vitro model. Fig. 4a shows cell viability of K562/ADR cells and PBMCs (lymphocytes population) after treatment with 10 μM of different TA samples for 48 h. It can be seen that TA has significant effect on cell viability of lymphocytes and K562/ADR cells, TAox has no toxic, while amino acid modified TA exhibit less toxic to lymphocytes, as compared to K562/ADR. In addition to PBMCs, hepatocytes are very important for toxicity concerns because TA is metabolized in the liver. AML-12 is a non-transformed mouse hepatocyte cell line that remains as an essential enzyme for TA metabolism. We preliminarily evaluated the effect of different TA samples on cell viability of AML-12 cells using MTT assay. Fig. 4b and 4c show cell viability of AML-12 after incubation with different TA samples for 48 h and 72 h, respectively. The results show that treatment with all TA samples exhibit low hepatotoxicity. Notably, unmodified TA exhibits toxicity in a concentration-dependent manner, but the modified ones were found to be less toxic to the cells, especially for high concentrations of the samples. According to dose–response curve fitting, The IC50 values of unmodified TA were approximately determined as 22.5 and 22.0 μM for 48 h and 72 h of incubation, respectively. For the modified TA samples, the IC50 values of most samples were not able to be determined, indicating that the IC50 values are beyond the tested concentration (>40 μM). These results suggest that modifications of TA structure by oxidation reaction and amino acid addition are able to prevent liver toxicity mediated by TA. These in vitro findings in toxicity concerns are considered beneficial for predictive information of further in vivo studies or clinical trials.

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Fig. 4

(a) Cell viability (cell counting) assay of K562/ADR and lymphocytes after treatment with 10 μM of different TA samples for 48 hrs (n = 2), (*p < 0.05, ns: not significant by unpaired t test). (b,c) Cell viability (MTT) assay of AML-12 cells after treatment with different TA samples for 48 hrs and 72 hrs, respectively (n = 3), (*p < 0.05, ns: not significant by one-way ANOVA).

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Although our cell viability analysis provides preliminary results on therapeutic potential of TA samples as an anti-cancer agent, it neither provides insight into the distribution of the cells in different cell cycle phases nor cell death mechanisms. As far as cell apoptosis and cell cycle disruption are concerned, cell cycle analysis by flow cytometry was carried out. The results demonstrate that TA is capable inducing cell death via apoptosis, in which 80% of cells were found in sub G0 phase (considered as cell apoptosis) at 24 h of induction and up to 90% for those of 48 h and 72 h, indicating a high efficiency for apoptosis induction of unmodified TA (Fig. 5). In cases of modified TA samples, they were found to induce an increasing cell population in the G2/M phase and Sub G0 phase, (but much lower than those observed in un-modified TA) at 24 h. As the time passed, the population of cells in G2/M phase was found to decrease with time, whereas those in Sub G0 phase were found to increase with time, suggesting the sequential events of G2/M arrest followed by apoptosis. In addition, the cell population in SSC/FSC graph (Figure S8) of the treated cells was found to shift to lower FSC (smaller size) and higher SSC (higher internal structure), compared to control. This change is considered as crucial cellular biophysical characteristic of apoptosis cell death. Thus, it can be concluded that the treated cells undergo apoptosis.

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Fig. 5

(a,b,c) Flow cytometric analysis of cell cycle distributions of K562/adr cell after treatment with different TA samples for 24, 48, and 72 hrs, respectively (n = 3). The final concentrations of TA, TA + Lys, TA + Phe and TA + Cys were 10 μM (TA equivalent), while that of TAox was 20 μM.

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It has been reported that a number of polyphenol compounds were capable of inducing cancer cell death via their capability to generate intracellular ROS (D'Angelo et al., 2017; Martino et al., 2019). Herein, we determined the level of ROS after incubation with different TA samples for different lengths of time. As the results show in Fig. 6a, all TA samples are found to increase the level of ROS within the cells after 24 h incubation, compared to untreated cells. Comparatively, unmodified TA seems to have more potent effect on ROS generation, compared to the modified ones. Surprisingly, the high-level ROS was maintained in un-modified TA treated when the incubation time had passed to 72 h, corresponding to apoptosis-inducing potency of TA, but those for modified TA samples were found to decrease (Fig. 6a). This would imply that having a capability to maintain a high level ROS is essential for inducing cell apoptosis by TA. It is important to notice that amino acid used in this experiment have no significant effect on intracellular ROS level (Figure S9). However, induction of cell apoptosis by the modified TA samples might occur through different ways. It is reported that polyphenol can induce autophagic apoptosis (Zhang et al., 2018) and autophagic cell death (Hasima and Ozpolat, 2014). In this context, lysosome has a crucial role in mediating cell death (Kavčič et al., 2017; Kroemer and Jäättelä, 2005). Therefore, we checked lysosome signals by acridine orange staining. For unmodified TA, AO fluorescence signals were found to slightly increase at 48 h of incubation. However, the signal was found to decrease to basal level after longer periods of incubation (72 h), indicating the end of the cellular degradation process (Fig. 6b). This phenomenon is a regular function of lysosomes where they can be activated for cellular degradation and clearance and return to the state of un-activation (basal level.) It should be noticed that low induction of lysosome mediation has no effect on cell apoptosis of TA, as observed from the above results. In contrast, there is a clear enhancement of AO signal (lysosomal signal) in a time-dependent manner in all modified TA samples. This result suggests that their actions in inducing cell death are totally different than un-modified TA. Possibly, death mechanisms would be mediated through over-activation of lysosomes such as autophagy-mediated apoptosis and autophagy cell death. This corresponds to the results from cell cycle analysis. These findings are of great interest in the context of cancer treatment, especially for apoptosis-resistant cancer cells. However, more studies need to be carried out.

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Fig. 6

(a) Flow cytometric analysis of intracellular ROS of K562/ADR cell after treatment with different TA samples for 24 hrs and 72 hrs (n = 3), (*p < 0.05, ns: not significant by unpaired t test). (b) Flow cytometric analysis of lysosome of K562/ADR cell after treatment with different TA samples for 24 hrs, 48 hrs, and 72 hrs (n = 3)., (*p < 0.05, ns: not significant by one-way ANOVA). The final concentrations of TA, TA + Lys, TA + Phe and TA + Cys were 10 μM (TA eqivalent), while that of TAox was 20 μM.

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