Ai-Ping Zhang, Yan-Ping Sun, Chemical Engineering Department, Taiyuan University of Technology, Taiyuan 030024, Shanxi Province, China
Supported by the National Natural Science Foundation of China, No.29776032 and the Natural Science Foundation of Shanxi Province, No.971013
Correspondence to: Yan-Ping Sun, Chemical Engineering Department, Taiyuan University of Technology, Taiyuan 030024, Shanxi Province, China.  ypsun@tyut.edu.cn
Telephone: +86-351-6010070
Received: 2003-10-10    Accepted: 2003-12-29

Abstract
AIM: To investigate the photocatalytic killing effect of photoexcited TiO₂ nanoparticles on human colon carcinoma cell line (Ls-174-t) and to study the mechanism underlying the action of photoexcited TiO₂ nanoparticles on malignant cells.

METHODS: Ls-174-t human colon carcinoma cells were cultured in RPMI 1640 medium supplemented with 199 mL/L calf serum in a humidified incubator with an atmosphere of 50 mL/L CO₂ at 37 °C. Viable cells in the samples were measured by using the MTT method. A GGZ-300 W high pressure Hg lamp with a maximum ultraviolet-A (UVA, 320-400 nm) irradiation peak at 365 nm was used as light source in the photocatalytic killing test.

RESULTS: The photocatalytic killing of Ls-174-t cells was carried out in vitro with TiO₂ nanoparticles. The killing effect was weak by using UVA irradiation without TiO₂ nanoparticles. In our studies, the photocatalytic killing effect was correlated with the concentration of TiO₂ and illumination time. Once TiO₂ was added, Ls-174-t cells were killed at a much higher rate. In the presence of 1 000 mg/mL TiO₂, 44% of cells were killed after 10 min of UVA irradiation, and 88% of cells were killed after 30 min of UVA irradiation.

CONCLUSION: When the concentration of TiO₂ is below 200 mg/mL, the photocatalytic killing effect on human colon carcinoma cells is almost the same as that of UVA irradiation alone. When the concentration of TiO₂ is above 200 mg/mL, the remarkable killing effect of photoexcited TiO₂ nanoparticles can be found.

Zhang AP, Sun YP. Photocatalytic killing effect of TiO₂ nanoparticles on Ls-174-t human colon carcinoma cells. World J Gastroenterol  2004; 10(21): 3191-3193
http://www.wjgnet.com/1007-9327/10/3191.asp

INTRODUCTION
The application of TiO₂ photocatalysis has received increasing attention since the first report of microbiocidal effects by Matsunaga et al. in 1985^[1]. In recent years, in contrast to many studies using TiO₂ powder for photodecomposition of organic pollutants^[2-9], few studies have investigated the application of TiO₂ in life science, especially in the field of cancer treatment^[10-12]. The incidence of colon cancer is rising in China. Despite that surgical operation is used currently, people have recognized its limitation. The way to treat cancer usually includes radiation therapy and chemical therapy, which may generate severe side effects in human body. Therefore, this study tried to investigate a new therapy for cancer. Ls-174-t cells were used as experiment objects in this study. The photocatalytic killing effect of TiO₂ nanoparticles on malignant cells and its killing mechanism were investigated.

MATERIALS AND METHODS
Reagent preparation
TiO₂ colloid solutions were prepared^[13,14] by hydrolysis of titanium isopropoxide, Ti [OCH (CH₃) ₂] ₄ (97%, Aldrich Chemical Co). In brief, 12.5 mL of Ti [OCH (CH₃) ₂]₄ was added to 2 mL isopropanol, then the mixture was added to 150 mL of distilled deionized water containing 2 mL of 700 mL/L nitric acid and vigorously stirred for 6 h at 75 °C. Approximately 150 mL of TiO₂ colloid solution being stable for several months at 4 °C was obtained after the organic layer was removed. The average diameter determined by Zetasizer 3000HSA (USA) was 21.2 nm.
     The pH value of TiO₂ colloid solutions used in the subsequent experiment had to be adjusted from 1.8 to 5.5-6.5 in order not to damage the normal growth of cells. Therefore, 1 mol/L NaOH aqueous solution and 1.5 mL/L polyvinyl alcohol were added to the colloid solutions before the pH adjustment to prevent the TiO₂ from precipitation. The final TiO₂ colloid solutions were sterilized by autoclaving and then diluted to the required concentration. Other chemical reagents used were all of analytical purity from commercial sources.

Cell culture and treatment
Human colon carcinoma cell lines Ls-174-t were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cell lines were cultured in RPMI 1640 (Gibco) medium supplemented with calf serum 100 ml/L, penicillin (100×10³ m/L) and streptomycin (100 mg/L). pH was maintained at 7.2-7.4 by equilibration with 50 mL/L  CO₂ . Temperature was maintained at 37 °C. Cells were sub-cultured with a mixture of ethylenedinitrile tetraacetic acid (EDTA) and trypsin. All experiments were performed using cells during the exponential growth phase. Cell concentration was determined by using a hemocytometer and the cell density was adjusted to the required final concentration.
      Ls-174-t cells were treated with TiO₂ diluted in RPMI 1640 medium for 2 h at 37 °C. Then the solutions were irradiated with a GGZ-300W high pressure Hg lamp (E_max = 365 nm) at room temperature. A UV pass filter was used to obtain a light wavelength between 300-400 nm. The light intensity at the liquid surface was measured by a VLX-3W radiometer-photometer (USA). The incident light intensity was 3.7 mW/cm². In our study, three groups of tests were carried out. One group was treated in the absence of TiO₂. Another group was treated in the absence of UVA. The third group was treated with different TiO₂ concentrations and irradiated by UVA.

Measurement of the viability of Ls-174-t cells
Viable cells in the samples were measured by using the MTT staining method^[15]. MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide] was dissolved in phosphate-buffered saline ( PBS, pH 7.4) at 5 mg/mL and filtered to be sterilized. Twenty microliters of stock MTT solution were added to all wells for an assay, and plates were incubated at 37 °C for 4 h. One hundred and fifty microliters of DMSO were added to all wells and mixed thoroughly to dissolve the blue-violet crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a Bio-Rad Novapath^tm microplate reader (Japan), using a test wavelength of 595 nm, taking the solution without MTT as control. Then optical absorptions [A] were obtained. Plates were normally read within 1 h after DMSO was added. The survival rate could be calculated according to [A]_t/[A]_i, where [A]_i is the optical absorption of untreated cells.

RESULTS
Determination of the average diameter of TiO₂ particles
Zetasizer 3000HSA (USA) was used to determine the average diameter of TiO₂ nanoparticles. The result is shown in Figure 1. The average volume size of TiO₂ nanoparticles was 21.2 nm.

Cytotoxicity of TiO₂ nanoparticles
The cytotoxicity of TiO₂ nanoparticles (without UVA irradiation) was determined by exposing cells to various concentrations of TiO₂ in RPMI 1640 (Gibco) medium for 24 h. The surviving fraction of the cells was greater than 90% when the concentration of TiO₂ was in the range of 1 000 mg/mL, as shown in Figure 2. The result confirmed that nonirradiated TiO₂ nanoparticles were not toxic to the Ls-174-t cells. It was consistent with those in literatures^[16-17].

Effect of photoexcited TiO₂ nanoparticles on Ls-174-t cells
The fact that the surviving fraction was greater than 90% after 30 min as shown in Figure 3 (A) indicated that TiO₂ nanoparticles without UVA irradiation showed little toxicity to living cells. The killing effect of UVA without TiO₂ is shown in (B) with the surviving fraction of Ls-174-t cells given as a function of the UVA light irradiation time. About 20% cells were killed after 30 min exposure, whereas after 20 min exposure, more than 90% of the cells survived. Once TiO₂ was added, the Ls-174-t cells were killed at a much higher rate as shown in (C). For example, in the presence of 1 000 mg/mL of TiO₂, 44% of the cells were killed after 10 min of UVA irradiation, and after 30 min irradiation, 80 % of the cells were killed. Therefore it was concluded that photoexcited TiO₂ nanoparticles had an active killing effect on Ls-174-cells.

Effect of TiO₂ concentration on Ls-174-t cells activity
The effect of the TiO₂ concentration ranging from 200 to 1 000 mg/mL on the rate of cell killing by UVA light is shown in Figure 4. The light intensity was 3.7 mW/cm² and kept constant. The experimental results demonstrated that cell viability decreased monotonically as TiO₂ concentration increased and cell viability decreased with time.
     Although a higher concentration of TiO₂ could achieve a higher reaction rate, the difficulties in separation and measurement had to be considered. So the effect of a higher TiO₂ concentration (C>1 029 mg/mL) on Ls-174-t cell activity was not investigated. The concentration of TiO₂ was set at <1 029 mg/mL with which the dark cytotoxicity was considered to be negligible. The range of TiO₂ concentrations was close to that used previously^[18].

Morphological changes of Ls-174-t cells
Untreated and TiO₂-treated cells were collected by centrifugation and resuspended in RPMI 1640 medium. The samples were pipetted into a 24-well plate, which was directly observed with an inverted phase-contrast microscope. When treated by photoexcited TiO₂, the cellular shape was condensed and nuclei were dispersed in fragments.

Figure 1(PDF) Volume size distribution of TiO₂.
Figure 2(PDF) After Ls-174-t cells were incubated in RPMI 1640 medium for 24 h without irradiation, the survival of Ls-174-t cells was shown.
Figure 3(PDF) Effect of light and TiO₂ on viability of Ls-174-t cells. (A) TiO₂ (1 000 mg/mL) in the dark; (B) no TiO₂ in the light; (C) TiO₂ (1 000 mg/mL) in the light. Initial cell concentration: 5×10⁵ cell/mL, light intensity: 3.7 mW/cm².
Figure 4(PDF) Influence of TiO₂ concentration on Ls-174-t cells activity. (A) 204 mg/mL, (B) 524 mg/mL, (C) 804 mg/mL, (D) 1 029 mg/mL.

DISCUSSION
In this experiment, the TiO₂ nanoparticle system was used, displaying its superiority. TiO₂ nanoparticles were easy to attach to the cellular membranes and accumulate. They were also easy to enter into the cytoplasm via phagocytosis^[19]. It could lead to accumulation of ROS on the surface of cell membranes and in the cytoplasm. Hence under light irradiation, TiO₂ nanoparticles had more significant cell killing effect in vitro.
      Human colon carcinoma cells treated with photoexcited TiO₂ nanoparticles (C>200 mg/mL) were effectively damaged, with cells contracted and a lot of cell fragments simultaneously observed by an inverted phase-contrast microscope^[18]. According to these characteristics, we assumed that the mechanism of photoexcited TiO₂ in killing human colon carcinoma cells might be through a series of oxidized chain reactions and in inducing cell death by reactive oxygen species^[20-25]. Human colon carcinoma cell damages occurred in two stages. The initial oxidative damage took place on the cell membranes, where the TiO₂ photocatalytic surface had its first contact with intact cells, the membranes became somewhat permeable. At this stage the cells did not lose their viability. Photocatalytic action made the cell membranes permeable, intracellular components began to leak from the cells and free TiO₂ nanoparticles might also diffuse into the damaged cells and directly attack intracellular components, eventually leading to cell death. It is different from the bactericidal effect of TiO₂ photocatalytic reaction. Bacteria are simple prokaryotic cells that do not contain the nucleus characteristics of eukaryotic cells. Whereas human colon carcinoma cells are eukaryotic cells and their structure is complex. Based on their structural differences, we assumed that killing cancer cells might be more difficult than killing bacteria by the photocatalytic reaction of TiO₂ nanoparticles.
      In the present study, cultured human colon carcinoma cells were effectively killed by photoexcited TiO₂ nanoparticles in virto. The concentration of TiO₂ affected the photocatalytic killing effect. When the concentration of TiO₂ was below 200 mg/mL, there was only a slight decrease in survival ratio after UVA irradiation for more than 30 min. It was almost the same as that of UVA irradiation alone. It indicated that minor cell membrane leakage might occur and the cell viability was not lost. When the concentration of TiO₂ was above 200 mg/mL, the survival ratio decreased rapidly with increasing TiO₂ concentration. It indicated that major rupture of cell membranes and decomposition of essential intracellular components might take place, thus accelerating cell death. It verified the mechanism of TiO₂ nanoparticles in killing human colon carcinoma cells.
      The photocatalytic killing effect of TiO₂ nanoparticles on human colon carcinoma cells suggested the idea of cancer treatment using TiO₂ nanoparticles and light irradiation. Under these conditions, it could be adapted to an anticancer modality by the local or regional treatment of the tumor with TiO₂ nanoparticles, followed by light irradiation focusing on the tumor. Although UVA light (320-400 nm) cannot penetrate the human body deeply, it may be possible that the modality will be applied to several human tumors in the future.

REFERENCES
1    Matsunaga T, Tomoda R, Nakajima T, Wake H. Photoelectro-chemical sterilization of microbial cells by semiconductor 
      powders. FEMS Microbiol Lett 1985; 29: 211-214
2    Byrne JA, Eggins BR, Brown NMD, Mckinney B, Rouse M. Immobilisation of TiO₂ powder for the treatment of polluted 
      water. Appl Catal B Environ 1998; 17: 25-36
3    Wang KH, Hsieh YH, Ko RC, Chang CY. Photocatalytic degradation of wastewater from manufactured fiber by titanium 
      dioxide suspensions in aqueous solution. Environ Int 1999; 25: 671-676
4    Byrne JA, Eggins BR, Byers W, Brown NMD. Photoelectrochemical cell for the combined photocatalytic oxidation of 
      organic pollutants and the recovery of metals from waste waters. Appl Catal B Environ 1999; 2: L85-L89
5    Horikoshi S, Satou Y, Hidaka H, Serpone N. Enhanced photocurrent generation and photooxidation of benzene sulfonate 
      in a continuous flow reactor using hybrid TiO₂ thin films immobilized on OTE electrodes. J Photochem Photobiol A 
      2001; 146: 109-119
6    Molinari R, Grande C, Drioli E, Palmisano L, Schiavello M. Photocatalytic membrane reactors for degradation of organic 
      pollutants in water. Catal Today 2001; 67: 273-279
7    Shephard GS, Stockenstrom S, de Villiers D, Engelbrecht WJ, Wessels GE. Degradation of microcystin toxins in a falling 
      film photocatalytic reactor with immobilized titanium dioxide catalyst. Water Res 2002; 36: 140-146
8    Muneer M, Singh HK, Bahnemann D. Semiconductor-mediated photocatalysed degradation of two selected priority 
      organic pollutants, benzidine and 1, 2-diphenylhydrazine, in aqueous suspension. Chemosphere 2002; 49: 193-203
9    Alhakimi G, Studnicki LH, Al-Ghazali M. Photocatalytic destruction of potassium hydrogen phthalate using TiO₂ and 
      sunlight: application for the treatment of industrial wastewater. J Photochem Photobiol A 2003; 154: 219-228
10  Fujishima A, Cai RX, Otsuki J, Hashimoto K, Itoh K, Yamashita T, Kubota Y. Biochemical application of 
      photoelectrochemistry: photokilling of malignant cells with TiO₂ powder. Electrochim Acta 1993; 38: 153-157
11  Mills A, Hunte SL. An overview of semiconductor photocatalysis. J Photochem Photobiol A 1997; 108: 1-35
12  Fujishima A, Rao TN, Tryk DA. Titanium dioxide photocatalysis. J Photochem Photobiol C 2000; 1: 1-21
13  Kormann C, Bahnemann DW, Hoffmann MR. Preparation and characterization of quantum-size titanium dioxide. J Phys 
      Chem 1988; 92: 5196-5201
14  O’Regan B, Moser J, Anderson M, Gratzel M. Vectorial electron injection into transparent semiconductor membranes 
      and electric field effects on the dynamics of light-induced charge separation. J Phys Chem 1990; 94: 8720-8726
15  Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. 
      J Immunol Methods 1983; 65: 55-63
16  Bernard BK, Osheroff MR, Hofmann A, Mennear JH. Toxicology and carcinogenesis studies of dietary titanium 
      dioxide-coated mica in male and female Fischer 344 rats. J Toxicol Environ Health 1990; 29: 417-429
17  Linnainmaa K, Kivipensas P, Vainio H. Toxicity and cytogenetic studies of ultrafine titanium dioxide in cultured rat liver 
      epithelial cells. Toxicol In vitro 1997; 11: 329-335
18  Xu MH, Huang NP, Xiao ZD, Lu ZH. Photoexcited TiO₂ nanoparticles through . OH- radicals induced malignant cells to 
      necrosis. Supramole Sci 1998; 5: 449-451
19  Cai RX, Hashimoto K, Itoh K, Kubota Y, Fujishima A. Photokilling of malignant cells with Ultrafine TiO₂ powder. Bull 
      Chem Soc Jpn 1991; 64: 1268-1273
20  Jaeger CD, Bard AJ. Spin trapping and electron spin resonance detection of radical intermediates in the 
      photodecomposition of water at TiO₂ particulate systems. J Phys Chem 1979; 83: 3146-3152
21  Rao MV, Rajeshwar K, Pal Verneker VR, DuBow J. Photosynthetic production of H₂ and H₂O₂ on semiconducting oxide 
      grains in aqueous solutions. J Phys Chem 1980; 84: 1987-1991
22  Harbour JR, Hair ML. Superoxide generation in the photolysis of aqueous cadmium sulfide dispersions. Detection by spin 
      trapping. J Phys Chem 1977; 81: 1791-1793
23  Harbour JR, Tromp J, Hair ML. Photogeneration of hydrogen peroxide in aqueous TiO₂ dispersions. Can J Chem 
      1985; 63: 204-208
24  Hong AP, Bahnemann DW, Hoffmann MR. Cobalt (II) tetrasulfop- hthalocyanine on titanium dioxide: a new efficient 
      electron relay for the photocatalytic formation and depletion of hydrogen peroxide in aqueous suspensions. J Phys 
      Chem 1987; 91: 2109-2117
25  Hidaka H, Horikoshi S, Serpone N, Knowland J. In vitro photochemical damage to DNA, RNA and their bases by an 
      inorganic sunscreen agent on exposure to UVA and UVB radiation. J Photochem Photobiol A 1997; 111: 205-213

   Edited by Zhang JZ and Wang XL Proofread by Zhu LH and Xu FM  

Reviews	Add
more>>