Confocal Raman spectroscopic analysis of the cytotoxic response to cisplatin in nasopharyngeal carcinoma cells

Abstract

Apoptosis of nasopharyngeal carcinoma cells (C666 cell line) induced by an anticancer drug cisplatin was investigated by confocal Raman micro-spectroscopy using near-infrared laser (785 nm) excitation in this study. The Raman spectra of C666 cells treated with different concentrations of cisplatin (0.5, 1, 5 and 10 μg mL⁻¹) for 24 h and different treatment times (6, 12, 18 and 24 h) with 5 μg mL⁻¹ cisplatin were collected separately. Difference in the intensities of Raman peaks assigned to the DNA band (783 cm⁻¹, 1338 cm⁻¹, 1523 cm⁻¹ and 1576 cm⁻¹) between the cells treated with cisplatin and control cells becomes greater as the concentration of cisplatin increases, indicating that the cytotoxicity of cisplatin for NPC cells is likely related to its concentration. The major difference between the apoptotic C666 cells incubated with cisplatin and the non-treated cells is the reduction in intensities of vibration bands generated by cellular nucleic acids, proteins and lipids. Large intensity reduction in nucleic vibrations at 783, 1523 and 1576 cm⁻¹ was observed upon apoptosis of the C666 cells. In particular, up to 14.1% and 49.6% reduction in the magnitude of the peaks at 783 cm⁻¹ and 1523 cm⁻¹ respectively in Raman spectra of the apoptotic cells was observed after 24 h of cisplatin treatment, which suggests the breakdown of phosphodiester bonds and DNA bases. Moreover, the intensity of peaks at 1002 and 1447 cm⁻¹ respectively fell to 40.9% and 43.1% of the original value, which indicates that cisplatin could induce apoptosis of C666 cells and reduce the amount of nucleic acid and protein in the cells. These results demonstrate that Raman spectroscopy is a novel, nondestructive mean for studying the anticancer-treated carcinoma cells, which could also provide abundant information about the changes in biochemical properties of cells and serve as an effective method for real time measurement of apoptosis.

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1 Introduction

For the design of new anticancer drugs, an in-depth understanding of the mechanisms underlying their biological effects is required. The mechanism of most anticancer drugs is that they can disorder biochemical and biophysical intracellular properties to induce apoptosis of the cancer cells.¹ It is therefore of interest to understand details of the molecular and biochemical mechanisms associated with the biological activity of existing commercial anticancer agents.

Nasopharyngeal carcinoma (NPC) has a remarkably distinctive ethnic and geographic distribution, which is a malignant tumour spreading in the whole world with much higher incidence in Chinese southern areas.² Of all the chemotherapy drugs, cisplatin is the most effective anticancer drugs widely used in NPC treatments. It is important to understand the pharmacokinetic mechanisms of cisplatin in NPC cells. Currently, the investigation of cancer cells treated with anticancer drugs in the in vitro culture has become one of the main test methods for assessing the efficacy of anti-cancer drugs. Moreover, monitoring the changes of intracellular substances plays a significant role in this method. Most current pharmacological, histological and cellular techniques, however, are complex procedures, or destruction to cell samples.³ Therefore, there is a need for new technology that provides a convenient and accurate method for detection of cancer cells treated with drugs.

Raman spectroscopy is an optical technique that depends on an energy shift due to the interaction with vibrational modes of molecules and can be used to detect changes of the structure and composition of substances at the molecular level.^4–7 The intracellular information about nucleic acid, protein and lipid contents as well as conformation can be probed by positions, intensities and line-widths of various spectral bands.^8–10 Therefore, Raman spectroscopy can differentiate between samples on the basis of the detectable changes in the spectral shape or intensity.^11,12 The potential of confocal Raman microspectroscopy for the analysis of biological tissue and the effect of external agents on the cell have been demonstrated.^13–16 Moreover, confocal Raman microspectroscopy is a noninvasive, highly sensitive, rapid technique and requires no sample labelling prior to analysis.^17,18 This technique has great potential to explore the sub-cellular biochemical structure as exemplified by its use in studies investigating the action of various agents on biological macromolecules as well as their interaction with cancer cells.^19,20

At present, Raman spectroscopy has emerged as a potential non-destructive diagnostic tool for many kinds of cancer cells, such as colon cancer, leukaemia and stomach cancer.^21,22 H. Yao et al. have applied near-infrared Raman spectroscopy to analyze the apoptosis of single human gastric cancer cells.²³ H. Nawaz et al. used Raman spectroscopy for prediction of the chemotherapeutic response to cisplatin in lung adenocarcinoma.²⁴ J. Lin et al. analysed the cytotoxic effect of cisplatin-treated leukemic cells by confocal Raman spectroscopy.²⁵ C. Cai et al. demonstrated the potential of Raman spectroscopy for identification and classification of normal human mononuclear cells and transformed cells.²⁶ And in previous studies, we have investigated the interactions of lymphoma cells with paclitaxel by the Raman technique.²² However, there is no clear recognition of the biochemical changes of cisplatin-treated NPC cells for different drug concentrations and different treatment times until now.

In this study, the cytotoxicity of cisplatin for the NPC cell line (C666) is explored by Raman spectroscopy. Spectral assignment and the changes of peak intensity were used to analyze the Raman spectra obtained from living cells treated with different concentrations of cisplatin (0.5, 1, 5 and 10 μg mL⁻¹) for 24 h and different treatment times (6, 12, 18 and 24 h) with 5 μg mL⁻¹ cisplatin separately. Spectra of cisplatin-treated and non-treated cells were also compared and analyzed. This work could provide abundant information about the changes in biochemical properties of cells treated with cisplatin and also provide useful evidence for clinical dosage optimization of cisplatin.

2 Materials and methods

2.1 Major drug reagents and instruments

Cisplatin injection (30 mg per 5 mL) was kindly provided by Fujian Provincial Tumour Hospital and cisplatin was kept in sterile PBS as a 0.5 mg mL⁻¹ stock solution. Hoechst 33258 (Hoechst Staining Kit, Beyotime) was stored at 1 mg mL⁻¹ in distilled water in a light-tight bottle at −20 °C. Just before use, a working solution was made by dilution of the stock solution to 0.5 μg mL⁻¹ in PBS (pH = 7.2). The working solution was held in and dispensed from a light-tight container. The other major reagents include fetal calf serum (HYCLONE), RPMI-1640 medium (GIBCO), and PBS (BOSTER). The major equipment used in this study include a CO₂ humidified incubator (THERMO) and a Renishaw micro-Raman spectrometer (InVia system).

2.2 Cell culture

Human nasopharyngeal carcinoma cells (C666 cell line) were obtained from the Fujian Provincial Tumour Hospital. C666 cells were grown in the RPMI 1640 medium (supplemented with 100 IU mL⁻¹ penicillin/streptomycin and 10% fetal calf serum, at pH 7.2) at 37 °C and 5% CO₂. The cells in the logarithmic growth phase were used as samples.

2.3 Cell sample preparation

2.3.1 Different treatment concentrations. The C666 cells were harvested from monolayer cultures by treatment with trypsin/EDTA solution, washed three times, centrifuged, and suspended in the RPMI 1640 medium with a density of 10⁵ cells per mL. The cell suspension was seeded in wells containing 1 × 10⁴ cells per mL on a microtiter plate (tissue culture grade, 12 wells, and flat bottom) on which they were grown to confluence. After 24 h, four samples of C666 cells were treated with 0.5, 1, 5 and 10 μg mL⁻¹ cisplatin for 24 h, respectively and the control group was supplied with an equal volume of RPMI 1640 medium. Before the measurement, the medium was removed, and the cells were trypsinized, centrifuged and rinsed three times in PBS to remove any residual medium.

2.3.2 Different treatment times. 24 h after the cells were seeded in wells, all excess culture media were removed and the C666 cells were washed with PBS solution two times. Finally, 1.98 mL RPMI 1640 medium and 20 μL cisplatin stock solution (0.5 mg mL⁻¹) were mixed thoroughly (5 μg mL⁻¹ cisplatin concentrations) and the mixture was added to each well containing cells. After cells were incubated with the cisplatin drug (5 μg mL⁻¹) for 6, 12, 18 and 24 h respectively, cell samples were chosen for Raman measurement. The control group was treated without cisplatin.

2.4 Detection of apoptosis

Parallel samples were prepared and the same cell samples were used for fluorescence staining and biochemical analysis. The C666 cells were collected and immediately placed in 75% alcohol for fixation. After washing with PBS, the cells were stained using Hoechst 33258 for 5 min. Then the cells were washed with PBS again and the fluorescence images were taken using a fluorescence microscope (Olympus IX71) with a 100× objective, using excitation at 350 nm and measuring emission at 460 nm.

2.5 Raman measurements of cells

A Renishaw InVia micro-Raman spectroscope with an ∼2λ spatial resolution, a 20 mW, 785 nm, semiconductor laser as an excitation source, was used for the collection of Raman spectra. Briefly, all wavelength-calibrated spectra were corrected for the wavelength dependence of a standard 520 cm⁻¹ vibrational band of a silicon wafer before Raman measurements. The microscope was operated under a 50× objective, which focused the laser beam onto a spot on the cell sample and the illumination pinhole was adjusted for the laser spot to cover the whole cell. The signal was integrated for 30 s and measured over a spectral range of 550 to 1750 cm⁻¹ with respect to the excitation frequency. We obtained three Raman spectra for one cell, and twenty individual living cells were selected from each sample for measurement. The Raman spectra of the cell associated with the autofluorescence background were displayed in computer in real time and saved for further analysis.

2.6 Data pre-processing

The raw spectra of C666 cells (control), C666 cells incubated with different cisplatin concentrations (0.5, 1, 5 and 10 μg mL⁻¹) for 24 h and C666 cells incubated for different treatment times (6, 12, 18 and 24 h) with 5 μg mL⁻¹ cisplatin in the 550–1750 cm⁻¹ range represented a combination of prominent cell auto-fluorescence, weak cell Raman scattering signals and noise. Thus, the raw spectra were pre-processed by adjacent five-point smoothing to reduce noise. And then, an automated algorithm for autofluorescence background removal was applied to the measured data to extract pure Raman spectra. The program was kindly offered by the BC Cancer Research Centre.²⁷ A fifth-order polynomial was found to be optimal for fitting the broad autofluorescence background in the noise-smoothed spectrum, and this polynomial was then subtracted from the spectrum to yield the cell Raman spectrum alone.²⁷ Each of background-subtracted Raman spectra was also normalized to the integrated area under the curve from 550 to 1750 cm⁻¹ to enable a better comparison of the spectral shapes and relative peak intensities among the different cell samples. After that, the spectra of the control and treated groups were respectively averaged over 20 cells that were measured.

3 Results and discussion

3.1 Biochemical analysis of C666 cells

To study the Raman spectra of apoptotic cells, it is essential to confirm cell apoptosis. C666 cells from the control group (treated without cisplatin) and the treatment group (treated with 0.5, 1, 5 and 10 μg mL⁻¹ cisplatin for 24 h) were observed by fluorescence microscopy after Hoechst 33258 staining. The basic process of apoptosis in vitro is based on the changes of chromatin and nucleolus distribution in the plasma membrane. The nuclei of cells from the control group were uniformly stained, which indicates that the cells had a complete structure. Compared with that, the chromatin of cells treated with 5 μg mL⁻¹ cisplatin for 24 hours was highly compacted and fragmented, demonstrating typical morphological changes of apoptosis occurred and most of the nuclei from the cells treated with 10 μg mL⁻¹ cisplatin for 24 hours were broken and densely stained.

3.2 Analysis of mean Raman spectra

In this study, we chose a laser excitation of 785 nm at a power of 20 mW and an irradiation time of 30 s for measuring the cell. There was less photodamage in cells at the near-infrared region.²⁵Fig. 1 shows the mean spectrum of the non-treated C666 cells from the control group (curve a, n = 20). The shaded areas represent the standard deviations of the means. Also shown at the bottom is the Raman spectrum of 5 μg mL⁻¹ cisplatin (curve b) measured under the same conditions. Here, the contrast between the mean Raman spectra of non-treated C666 cells and 5 μg mL⁻¹ cisplatin illustrates that the drug almost has no interference in the Raman signal of the cell, which ensures the accuracy of Raman spectral analysis. As shown in Fig. 1, high quality cell Raman spectra are obtained, containing a wealth of intracellular signals, characterized by Raman peaks based on the specific DNA backbone, DNA ring base and protein vibrations. Table 1 lists tentative assignments for the observed Raman bands, according to the literature data.^{2,3,20,23,24,28}

Fig. 1 Mean Raman spectrum ± SD for the non-treated C666 cells (curve a) and the Raman spectrum of the 5 μg mL⁻¹ cisplatin (curve b).

Table 1 The peak positions and tentative assignment of Raman spectra^a

Raman shift (cm^−1)	Tentative assignment
a Abbreviations indicate: A, adenine; G, guanine; C, cytosine; T, thymine; U, uracil; ν, stretching; δ, deformation; γT, twisting; γW, wagging; sym., symmetrical.
602	Ribose phosphate
640	Tyrosine: ring breathing
718	A, C, T, U: ring breathing,
783	ν(OPO) sym. of phosphodiester
865	Ribose: ν(CC), ring breathing, ν(COC)
938	a-helix: ν(CC) skeletal
957	Membrane protein: C–C skeletal β-sheets
1002	Phenylalanine: ring breathing
1031	Phenylalanine: δ(CH)
1064	Lipids: ν(CC) skeletal
1081	Lipid: ν(CC) skeletal of acyl backbone
1127	Proline: ν(CN)
1157	Protein: ν(CC, CN)
1208	Nucleotides: base ν(CN), Tyr, Phe
1315	Proteins: ring ν/γT(CH2, CH3)
1338	Polynucleotide chain
1447	Lipids, proteins: δ(CH2)
1523	C
1576	A, G
1656	Amide-I: ν(CO)

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3.3 Spectral features of C666 cells treated with cisplatin for different concentrations

Fig. 2 shows the mean Raman spectra of C666 cells (control) and cells treated with different concentrations of cisplatin (0.5, 1, 5 and 10 μg mL⁻¹) for 24 h (spectra line a–d). All spectra exhibit similar overall features, characterized by Raman peaks based on the specific DNA backbone, DNA ring base, lipid, amino acid, and protein vibrations.

Fig. 2 Mean Raman spectra of C666 cells treated without cisplatin (Ctrl) and incubated with different concentrations of cisplatin ((a) 0.5 μg mL⁻¹, (b) 1 μg mL⁻¹, (c) 5 μg mL⁻¹ and (d) 10 μg mL⁻¹) for 24 h.

As DNA is the major target of cisplatin,³ the Raman peaks assigned to DNA–purine bases and the DNA backbone (783 cm⁻¹, 1338 cm⁻¹, 1523 cm⁻¹ and 1576 cm⁻¹ as mentioned in Table 1) were selected to analyze the changes of nucleic acids in C666 cells treated with various concentrations of cisplatin. As the concentration of cisplatin increases, the band at 783 cm⁻¹ attributed to ring breathing of cytosine/thymine/uracil and O–P–O symmetric stretch of the phosphodiester bond in DNA gradually decreases in intensity and shifts slightly to 790 cm⁻¹, indicating that the content and the double helix structure of DNA have been changed possibly. The band at 1338 cm⁻¹ is attributed to the polynucleotide chain (DNA–purine bases). The intensity of this peak decreases, however, its position does not change as the concentration of cisplatin increases, suggesting that changes occur only in the content of DNA rather than the double helix structure of it. Meanwhile, the peaks at 1523 cm⁻¹ and 1576 cm⁻¹ due to cytosine, adenine and guanine of DNA also decrease obviously as the concentration of cisplatin increases, which illustrates that DNA replication has been affected resulting from the destruction of base pairs of DNA.

The biochemical effects of DNA treated with cisplatin at different concentrations are evaluated quantitatively by normalized Raman intensity. For each experiment (one control group and four different concentration treated groups), Raman measurements were performed on twenty individual living cells and three spectra were acquired from each cell by repeated measurements. Therefore, in total sixty Raman spectra were obtained for each experimental group after the removal of the fluorescence background from the original data. While for every spectrum, we calculated the total integrated area under the curve from 550 to 1750 cm⁻¹. Then the integrated intensity was divided by the intensities of Raman peaks obtained from cells to generate a standardized “normalized Raman intensity”. This reduces the spectral intensity variations between different spectra and facilitates more accurate spectral shape analysis.^29–31

Fig. 3 shows a comparison of the mean intensities and standard deviations of the selected peaks assigned to DNA (783 cm⁻¹, 1338 cm⁻¹, 1523 cm⁻¹ and 1576 cm⁻¹ as mentioned above) with significant differences (Student's t-test analysis) between the control group and four experimental groups treated with different concentrations. As shown in Fig. 3, p < 0.05 was labeled as *. The Student's t-test was performed using the SPSS software package (SPSS Inc., Chicago).

Fig. 3 Comparison of the mean intensities and standard deviations of the selected peaks assigned to DNA (783 cm⁻¹, 1338 cm⁻¹, 1523 cm⁻¹ and 1576 cm⁻¹) with significant differences (Student's t-test analysis) between the control group and four experimental groups treated with different concentrations. p < 0.05 was labeled as *.

Motivated by this, it is evident that the differences in the mean Raman intensities of selected DNA bands between the cells treated with cisplatin and control cells become greater as the concentration of cisplatin increases, which indicates that there is something abnormal in the structure and the content of biochemical molecules inside tumor cells and most of the cells attain apoptosis or death with the increase in the concentration of the drug. Thus, we conclude that the cytotoxicity of cisplatin for C666 cells is likely related to its concentration, which is in agreement with the analysis of spectra above. This result is in accordance with some reports on cisplatin-based programmed cell death (apoptosis). Cisplatin is the abbreviation of cis-diammine-dichloroplatinum(II), and its molecular formula is cis-Pt(NH₃)₂Cl₂.²⁵ Cisplatin has clinically useful antitumour properties and is a well-established chemotherapeutic agent with a known mode of action. Recent studies have shown that apoptosis occurred in cisplatin-treated cells.³ In the nucleus, it binds with DNA forming inter-strand and intra-strand crosslinks which lead to cell cycle arrest and apoptosis. The formation of inter-strand and intra-strand crosslinks between cisplatin and DNA leads to conformational changes of the DNA.²⁴ Therefore, the main changes of the spectral shape from the control cells and the apoptotic cells treated with different concentrations of cisplatin observed are the reduction in intensities of vibration bands and the slight shift of Raman bands generated by cellular nucleic acids.

3.4 Spectral features of C666 cells treated with cisplatin for different times

Fig. 4 shows the mean Raman spectra of C666 cells treated without cisplatin (spectra line Ctrl), and those treated with 5 μg mL⁻¹ cisplatin for 6, 12, 18 and 24 h (spectral line a–d), respectively. The major changes observed are labelled. Comparison of five spectra shown in Fig. 4 suggests that the conformation and position of peaks are similar, however their intensities change with the increasing time of treatment. In Fig. 4, as the cisplatin-treatment time increases, the band at 783 cm⁻¹ assigned to ring breathing of cytosine/thymine/uracil and O–P–O symmetric stretch of the phosphodiester bond in DNA also decreases in intensity, which indicates that the conformation of DNA has been changed possibly. The peaks at 1523 cm⁻¹ due to cytosine and at 1576 cm⁻¹ due to adenine/guanine of DNA also decrease obviously, which illustrates that DNA replication has been affected, resulting from the destruction of base pairs of DNA. The decrease of the intensity of Raman peaks may result from covalent bonding of cisplatin and DNA in C666 cells,³ and this result is consistent with other literature.²⁴ The cisplatin reaction denatures the double helix by covalent bonding, breaking the Watson–Crick base pairs.²⁵ When cisplatin passively diffuses across the cell membrane, cisplatin will bind to the N-7 position of guanine in DNA, which weakens the hydrogen bonding of the G–C base pair, resulting in unwinding of the double helix ultimately.²⁶

Fig. 4 Mean Raman spectra of C666 cells treated without cisplatin (Control) and incubated with 5 μg mL⁻¹ cisplatin at different times: (a) 6 h, (b) 12 h, (c) 18 h, and (d) 24 h.

In addition, the Raman bands at 1002 cm⁻¹ and 1031 cm⁻¹ assigned, respectively, to the C–C ring breathing and C–H deformation of phenylalanine both decrease with the increase in the treatment time of the agent, which reflects the changes in amino acid of the protein molecule. Moreover, the peak at 1447 cm⁻¹ is assigned to the CH₂ deformation mode of proteins or lipids and the bands at 1656 cm⁻¹ are characteristic stretching vibrations of C=O of Amide I in general. And their intensity decreases as the treatment time increases, which indicates that the double helical structure of the protein has been changed possibly.²⁴ With the longer treatment time, the aggregated active materials in C666 cells are distorted and denatured more and more severely and their active mechanisms are disrupted absolutely.²⁵ In brief, those changes will cause disorder for the structure of proteins and the fierce loss of cell activity leads to the decrease of Raman intensity.

The biochemical and physiological effects of NPC cells treated with cisplatin at different times are also evaluated quantitatively by normalized Raman intensity. With these normalized data processing applied for the four different treatment time groups and the control group, we obtained a comparison graph (Fig. 5) of the Raman intensity for DNA bases (at 783 cm⁻¹ and 1523 cm⁻¹) and proteins (at 1002 cm⁻¹ and 1447 cm⁻¹).

Fig. 5 Changes in the Raman bands of C666 cells treated with 5 μg mL⁻¹ cisplatin for 0, 6, 12, 18, and 24 h at (A) 783 cm⁻¹, (B) 1523 cm⁻¹, (C) 1002 cm⁻¹ and (D) 1447 cm⁻¹.

As shown in Fig. 5A, the peak intensity at 783 cm⁻¹ assigned in the mode of ring breathing of cytosine, thymine, and uracil and the O–P–O symmetric stretching vibration of the phosphodiester bond in DNA significantly decrease with a greater degree due to the binding of cisplatin as the treatment time increases.²⁵ The intensity of the peak at 783 cm⁻¹ fell to 85.9% after 24 h treatment. The intensity at 783 cm⁻¹ can be representative of the content of cytosine, thymine and uracil, and the conformation of the phosphodiester bond. This could be indicative of the induction of conformational changes in the form of DNA due to the formation of intra-strand crosslinking of cisplatin with cytosine and binding with thymine.²⁴ This represents a Raman marker of the binding of cisplatin with the DNA bases. Moreover, the peak intensity at 1523 cm⁻¹ associated with cytosine is approximately reduced to 50.4% compared with the control (Fig. 5B). These results demonstrate that longer drug treatment time leads to more serious damage to the integrity of DNA and the decrease of nucleic acid concentration.²⁵ It has been shown that endogenous endonuclease activity is a major factor in the process of apoptosis, resulting in the DNA cleavage and nucleolus segregation and dispersal. The increasing DNA degradation and membrane permeability could be observed in terminal apoptotic cells, and the degraded nucleic acid diffused through the cell membrane, which caused a significant loss of nucleic acids.²⁶

Changes in the bands at 1002 cm⁻¹ (C–C ring breathing of the phenylalanine) and 1447 cm⁻¹ (CH₂ bending mode of proteins) are related to the protein structure as shown in Fig. 5C and D. As the treatment time increases, the intensity of the 1002 cm⁻¹ band becomes lower and is reduced to 40.8% after 24 h treatment, while the intensity of the 1447 cm⁻¹ band decreases to 43.1%, compared with the control, which shows that the intracellular protein content decreases continually due to drug treatment. This could be further confirmed by the reduction of intensity of other protein-related peaks such as 1656 cm⁻¹. The reduction of the intracellular protein content might be attributed to the decreased protein synthesis and increased protein hydrolysis.²⁵ This may be due to the fact that after the conformational changes caused in the DNA by cisplatin binding, cisplatin inhibited the proliferation of tumour cells, resulting in decreasing cell proliferation-related protein expression and weakening protein anabolism.³² On the other hand, the apoptotic process was accompanied by the hydrolysis of certain proteins,³³ for example activation of caspases during apoptosis resulted in the hydrolysis of critical cellular substrates.³⁴

4 Conclusion

In this study, confocal micro-Raman spectroscopy was used to study the interactional efficacy of nasopharyngeal carcinoma cells (C666 cell line) in vitro with cisplatin. Raman spectra of individual living C666 cells and those treated with different concentrations of cisplatin (0.5, 1, 5 and 10 μg mL⁻¹) for 24 h and different treatment times (6, 12, 18 and 24 h) with 5 μg mL⁻¹ cisplatin were successfully obtained to detect the changes of the biomolecular structure and the content during the process of apoptosis. Difference in the intensities of Raman peaks assigned to the DNA band (783 cm⁻¹, 1338 cm⁻¹, 1523 cm⁻¹ and 1576 cm⁻¹) between the cells treated with cisplatin and control cells becomes greater as the concentration of cisplatin increases, indicating that the cytotoxicity of cisplatin for NPC cells is likely related to its concentration. Specific biomolecular differences were observed including the decrease in the cellular DNA concentration and the change in the protein structure as the interactional time of cells and cisplatin increases, depending on the analysis of spectral assignment and the changes of peak intensity. These results suggest that the cytotoxicity of cisplatin for the C666 cells would increase gradually with increasing concentration and treatment time and may provide useful evidence for clinical dosage optimization of cisplatin. This study also demonstrate that Raman spectroscopy could provide abundant information about the changes in biochemical properties of cells and serve as a novel, nondestructive, effective method for real time measurement of cell apoptosis.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 11104030), the Science and Technology Project of Fujian Province (no. 2012J05004), the Project of the Educational Department of Fujian Province (nos JA10171 and JA11055) and the Developmental Fund of CHEN Ke-ji Integrative Medicine (no. CKJ2011010).

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