Hao
Huang
*a,
Hong
Shi
a,
Shangyuan
Feng
*b,
Weiwei
Chen
a,
Yun
Yu
a,
Duo
Lin
a and
Rong
Chen
b
aCollege of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, China. E-mail: cfjtcm@126.com; Tel: +86 591 22861151
bKey Laboratory of OptoElectronic Science and Technology for Medicine, Ministry of Education, Fujian Normal University, Fuzhou, Fujian 350007, China. E-mail: syfeng@fjnu.edu.cn; Tel: +86 591 83489919
First published on 5th November 2012
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.
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.
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). |
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) |
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.
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 CO 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
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