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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 3  |  Page : 1144-1152

Cellular and spectroscopic characterization of cancer stem cell-like cells derived from A549 lung carcinoma


1 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
2 Chemistry Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
3 Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Web Publication4-Jan-2017

Correspondence Address:
Badri N Pandey
Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai - 400 085, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.171365

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 > Abstract 

Background: Cancer stem cells (CSCs) are increasingly being realized to play a significant role in the mechanism of chemo-, radio-resistance, and metastasis of cancer. However, studies for spectral markers of CSCs using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy are limited in the literature.
Materials and Methods: In the present study, CSCs obtained from single cell assay of human lung adenocarcinoma (A549) cells were characterized using CD44+/CD24−/low phenotype expression, Hoechst 33342 dye efflux assay, and expression of stemness genes. Spectral changes in cancer cells and clones enriched with CSCs were studied by FT-IR and CD spectroscopy.
Results: The changes in FT-IR spectra of clones enriched with CSCs showed the difference in the secondary protein structure as compared to nonstem cancer cells. Moreover, A549 clone cells showed higher C-O band of carbohydrates and deoxyribose ring vibrations of Z-form of DNA. These results were further corroborated with CD spectroscopy that showed increased alpha helix proteins and difference in DNA conformation in clones enriched with CSCs. FT-IR studies also showed higher imidazole-metal interactions in clones enriched with CSCs. These results are in agreement with higher activity of one of the metalloproteins that is, superoxide dismutase in clones enriched with CSCs and their increased radioresistance.
Conclusions and General Significance: Overall, these observations provide novel FT-IR and CD spectroscopy signatures in A549 clones enriched with CSCs, which may have implications in the quantifying magnitude of CSCs as prognostic markers in cancer therapy.

Keywords: Cancer stem cells, lung cancer, radiosensitivity, single cell assay, spectroscopy


How to cite this article:
Balla MM, Ningthoujam RS, Kumar M, Bandekar JR, Pandey BN. Cellular and spectroscopic characterization of cancer stem cell-like cells derived from A549 lung carcinoma. J Can Res Ther 2016;12:1144-52

How to cite this URL:
Balla MM, Ningthoujam RS, Kumar M, Bandekar JR, Pandey BN. Cellular and spectroscopic characterization of cancer stem cell-like cells derived from A549 lung carcinoma. J Can Res Ther [serial online] 2016 [cited 2017 Oct 18];12:1144-52. Available from: http://www.cancerjournal.net/text.asp?2016/12/3/1144/171365


 > Introduction Top


Tumors consist of a subpopulation of cells known as “cancer stem cells” (CSCs) which are suggested to be involved in metastasis and resistance against therapeutic agents.[1],[2],[3],[4],[5] CSCs have higher ability to survive after chemo- and radio-therapy and thus considered as a major cause of tumor recurrence. Hence, understanding the CSC biology is currently getting emphasized for improved cancer therapy.

The fraction of CSCs in human tumor mass is low (0.5–5%) and vary across the tumor types and their clinical stages.[1],[6],[7] CSCs are generally characterized by expression of cell surface markers and tumor-forming ability in severe combined immune deficient mice.[1] These assays have been exploited to characterize CSCs in leukemia, breast, brain, colon, and other cancer types.[1],[8] However, these techniques are very expensive involving complicated, invasive procedures to evaluate the rare CSCs of particular tumor type. The available markers for CSCs are CD44+/CD24, CD133, ESA, P63, ABCG2, CD20, CD90, and Hoechst 33342low/side population cells.[8],[9],[10],[11],[12] These markers are not common across the tumor types, and their magnitude of expression varies in patients. In principle, CSCs would exhibit common properties in various tumor types which can be assessed by the techniques, such as Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy.

Quantitative knowledge about CSCs in a tumor tissue could be exploited for prognosis of metastasis and recurrence after cancer radio- and chemo-therapy.[13],[14] In this regard, FT-IR micro-spectroscopy has been used to differentiate the invasive prostate cancer tumor samples [15] which provides further hope to utilize such techniques in the clinical scenario. The changes in embryonic [16] and adult stem cells [17],[18] have been studied using FT-IR spectroscopy. However, FT-IR studies related to CSCs are limited to renal and esophageal adenocarcinoma cell lines showing changes in lipid, phospho-diester, and glycogen band in CSCs.[19],[20] It may be worthy to mention that CSCs in these studies are obtained either by fluorescence dye based sorting or by sphere culture. Usefulness of fluorescence based sorting technique has limitation of wear and tear of cells. Moreover, stem cells are sensitive to photo-induced free radicals such as singlet oxygen produced during fluorescence based sorting, which may affect cell surface chemistry required for stem cell quantification.[21],[22] The heterogeneity in cell population obtained from sphere culture may not truly represent the stem cells.[23] When compared to these studies, we have employed single cell assay (SCA) in which single cells (plated in each well of 96 well plate) forming the clone were considered to be CSCs. This assay is considered to be “gold standard procedure” to obtain the stem cells in vitro in which stem cells are characterized based on their self-renewal ability.[24],[25] Furthermore, A549 clones and cancer cells were characterized using CD44+/CD24−/low phenotype expression, the Hoechst 33342 dye efflux method, and expression of stemness genes, such as OCT4, NANOG, and glycine decarboxylase (GDC). Hoechst 33342 dye efflux method is used to characterize CSCs based on the dye efflux by transporters.[26] Using FT-IR studies, we have shown imidazole metal interactions in human lung adenocarcinoma clones enriched with CSCs, which was further corroborated with their increased superoxide dismutase (SOD) activity and radio-resistance. Furthermore, we have validated our FT-IR observation about conformational changes in proteins/nucleic acids of clones enriched with CSCs using CD spectroscopy.


 > Materials and Methods Top


Cell culture

A549 lung adenocarcinoma cell line was obtained from National Center for Cell Sciences, Pune, India. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, USA) supplemented with 10% fetal calf serum (FCS; Hi-Media, Mumbai, India) and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). Cultures were maintained in incubator at 37°C in 5% CO2 atmosphere.

Single cell assay

SCA of A549 cells was carried out as described previously with some modifications.[24] Briefly, at 80% confluency, A549 cultures were harvested by trypsinization, washed and cells were re-suspended in the complete medium. Cells were serially diluted in complete medium up to 1000 cells/mL and 300 μl of this stock was suspended in 30 mL of medium. In each well of 96-well plate, 100 μl of cell suspension was dispensed to obtain single cell per well, and wells were randomly checked (at least 20 wells) under a microscope to confirm single cell in each well. The plate was incubated in culture conditions for 12 days. Cells were harvested by trypsinization, and cells from different wells were pooled to obtain desired cell number for further experiments. These cells will be referred as “A549 clone cells.” Another group of cells were obtained from routine A549 culture at 80–90% confluency and will be referred as “A549 cancer cells.” Wherever required, cell viability was determined using trypan blue dye exclusion assay and counted using a hemocytometer.

Immunocytochemistry of CD44/CD24

About 1000 cells obtained either from A549 cancer cells or from individual clones were plated on glass cover slip for overnight. After that, culture medium was removed; cells were washed with prewarmed phosphate-buffered saline (PBS) and fixed using ice-cold acetone for 10 min. The fixed cells were again washed with PBS thrice. Cells were incubated with PBS containing 1% bovine serum albumin for 30 min at room temperature followed by 30 min incubation with CD44-FITC and CD24-PE antibodies (1:1; dilution 1:200, BD, Pharmingen, USA). Subsequently, cells were washed thrice with PBS, and the samples were mounted with antifade reagent (Molecular Probes, USA) and observed using confocal microscopy (LSM510 Meta, Carl Ziess, Germany).

Hoechst 33342 dye efflux method

A549 clone and cancer cells were subjected to Hoechst 33342 dye efflux assay as mentioned previously.[12] Harvested cells were washed with PBS followed by suspending them in complete medium and plating on coverslip for overnight. Attached cells on coverslip were labeled (at 37°C for 90 min) with Hoechst 33342 (Sigma; USA, 5 μg/mL) ±50 μM verapamil (Sigma). The dye with or without inhibitor was prepared in DMEM supplemented with 2% FCS and 1mM HEPES. After labeling, cells were washed with medium at 4°C and images were acquired using confocal microscope (excitation with 364 nm ultra violet [UV] laser and emission at 540–580 nm). Using these images, fluorescence intensities of cells were calculated using NIH image J software (Research Services Branch, NIMH, Bethesda, USA) for 300 cells each for A549 cancer and clones. Mean fluorescence intensity/per cell was expressed. From these images, Hoechst 33342−/low cells were also counted for 300 cells each for A549 cancer and clones. The cells showing fluorescence intensity lower than mean fluorescence intensity of that particular group were considered Hoechst 33342−/low cells.

Reverse transcriptase polymerase chain reaction

Total RNA was isolated from A549 clones and cancer cells using TRI reagent (Sigma-Aldrich, USA) and quantified by using Nanodrop 2000 (Thermo Scientific, USA). One micro gram of total RNA was reverse transcribed using reagents from superscript vilo cDNA synthesis kit (Life Technologies Corp., USA). GAPDH was used as an internal control and reaction mixture without template as a negative control. Gene specific primers for human OCT4, NANOG, and GDC were used and listed in [Table 1]. The polymerase chain reaction (PCR) conditions for human OCT4, NANOG, and GDC were 1 cycle of 5 min at 94°C; 32 cycles of 1 min at 94°C, 30 s at 57°C, and 1 min at 72°C; and 1 cycle of 5 min at 72°C. GAPDH was amplified under similar conditions for 25 cycles.
Table 1: Primer sequences and product sizes of the genes used

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Fourier transform infrared spectroscopy

A549 cancer or clone cells obtained after trypsinization were washed and fixed in 4% paraformaldehyde. Cells were centrifuged and suspended in 0.9% saline to get 100,000 cells per μl. For FT-IR spectroscopy, 2 μl of cell suspension was placed on CaF2 window and dried at 37°C for 30 min followed by vacuum drying at room temperature for ~30 min to eliminate excess water. Absorption spectra were acquired (400–4000 cm −1) in the transmittance mode using FT-IR spectrometer and then converted to absorption spectrum (BOHMEM, 4 cm −1 spectral resolution, 500 scans to obtain high signal/noise ratio). Obtained spectra were corrected by subtracting the saline spectrum. The second derivative of the spectra was obtained after 13-point smoothing by Savitzky-Golay method (3rd polynomial, 13 smoothing points)[16] using Originpro 8.0 (Origin Lab, USA) software. Three independent experiments were carried out and values (mean ± standard deviation) of selected ranges of wave numbers were plotted. Typical representative spectra for these ranges were also shown in [Supplementary Figure S1]. Wave numbers of respective bond vibrations and their references have been listed in [Table 2].
Table 2: Assignment of bond vibrations in FT-IR spectroscopy

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Circular dichroism spectroscopy

Circular dichroism spectra were recorded on Jasco J-815 CD spectrometer. Cells were washed with PBS (10 mM; pH 7.5) and suspended. CD spectra of cells (3 × 105 cells in 100 μl of PBS) were recorded in a 0.1 cm path length quartz cuvette at room temperature (20°C). Each spectrum was recorded for the wavelength range of 200–325 nm (scan rate: 50 nm/min, bandwidth: 1 nm, data pitch: 0.5 nm, response time: 1 s). Three consecutive accumulations were recorded for each sample to obtain the average spectrum. All spectra were corrected by subtracting the background spectrum of PBS.

Superoxide dismutase assay

SOD enzyme activity was performed in A549 cancer and clone cells following epinephrine method.[32] In brief, cells were harvested by trypsinization followed by washing with ice-cold PBS. Cell lysates were prepared using cell lysis buffer (Sigma) and protein concentration of lysates was determined using Bio-Rad DC protein assay kit. Equal amount of proteins were used for SOD assay and enzyme activity was expressed in terms of U/mg protein.

Irradiation and clonogenic assay

A549 cancer and clone cells were seeded (300 cells per 60 mm culture dish [BD Bioscience]) for overnight in 4 ml DMEM containing 10% serum. Subsequently, cells were g-irradiated (2 and 6 Gy;60 Co teletherapy Bhabhatron-II, Panacea Medical Technologies, Bengaluru, India; dose rate: 1 Gy/min) and were cultured for 12 days. Then colonies were washed with PBS, fixed with 70% ethanol, and stained with 0.5% crystal violet. Colonies were counted and survival fraction was calculated as mentioned earlier.[33]

Statistical analysis

Unless mentioned, mean are values out of three independent experiments and error bars are standard deviation. Where ever required, statistical analysis was performed using Originpro 8.0 (Origin Lab, USA) software. Student's t-test was used for comparing the means of two groups. Unless mentioned, values were considered significantly different at P < 0.05.


 > Results Top


Single cell assay of A549 cells and characterization of A549 clones

The percentage of clones in A549 cell line was evaluated by SCA. Clone-forming cells are 33.3 ± 7.3% of total A549 cancer cells (representative image of clone was shown in [Figure 1]a). When compared to cancer cells, CSCs are known to have higher expression of CD44 and lower expression of CD24 (i.e., CD44+/CD24−/low).[8],[11] Using confocal microscopy, the A549 clones and cancer cells were further characterized for the expression of CD44 and CD24 [Figure 1]b. A549 clones showed higher fluorescence of CD44 as compared to the cancer cells. The images were further quantified for the fraction of CD44+/CD24−/low in A549 cancer and clone cells [Figure 1]c. The fraction of cells showing CD44+/CD24−/low was significantly higher (P < 0.001) in A549 clones (~81%) compared to cancer cells (~25%) suggesting enrichment of cells expressing CSCs marker in clones.
Figure 1: (a) A549 single cell clone at 10× magnification, (b) immunocytochemistry of A549 cancer cells and clones for CD44 and CD24 expression, (c) the images were acquired and number of CD44+ and CD24−/low were counted in different fields for total 300 cells. Graphical representation of percentage cells positive for CD44+ and CD24−/low in A549 cancer cells and clones. *Significantly different when compared to A549 cancer cells (P < 0.001)

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The clone cells obtained from SCA was further evaluated for their stem cell property using a well-established Hoechst 33342 dye efflux method [Figure 2]a,[Figure 2]b,[Figure 2]c, which is based on efflux of dye due to higher expression of ATP-binding cassette G2 family (ABCG2) transporter proteins in CSCs. Confocal microscopic examination of A549 cancer and clone cells labeled with Hoechst 33342 dye showed more number of low fluorescence cells in A549 clones suggesting their dye effluxing ability [Figure 2]a i-iv. To further establish the specificity of dye efflux through ABCG2 transporters in clones, samples were treated with verapamil, a blocker of ABCG2 transporters. Treatment with verapamil resulted in the reversal of fluorescence intensity in A549 clone cells [Figure 2]a v-viii confirming their CSCs property. Images were further quantified for the mean fluorescence intensity per cell (MFI/cell) in A549 cancer and clone cells without or with verapamil treatment [Figure 2]b. Compared to A549 cancer cells, MFI/cells was lower (~45%) for clones, which however, significantly (P < 0.001) reverted when cells were treated with verapamil. It was interesting to note that, after verapamil treatment, the recovery of fluorescence intensity was higher (~75%) in A549 clone compared to the cancer cells (~20%). The fraction of Hoechst 33342low cells in A549 clone was significantly higher (P < 0.001; ~66%) compared to cancer cells (~16%) as determined from the confocal microscopy images [Figure 2]c. Taken together, these results confirm the higher fraction (enrichment) of CSCs in A549 clones obtained from SCA as compared to A549 cancer cells.
Figure 2: (a) Fluorescence images of Hoechst dye labeled A549 cancer and clone cells without (i-iv) or with verapamil (v-viii) treatment by confocal microscopy at 40× magnification magnification; arrows show cells with low fluorescence and dye efflux, (b) mean fluorescent intensity per cell in A549 cancer and clone cells; *significantly different when compared to cells without verapamil treatment (P < 0.001), (c) Percentage of Hoechst 33342−/low in A549 cancer cells and clones. *Significantly different when compared to A549 cancer cells (P < 0.001)

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To further confirm the stemness, the expression of OCT4, NANOG, and GDC genes were studied in A549 clones and cancer cells. Higher activity of GDC in lung cancer tumor-initiating cells has been shown previously.[34] A higher expression of stem cell marker genes OCT4, NANOG, and GDC was observed in A549 clones as compared to A549 cancer cells [Figure 3].
Figure 3: Reverse transcriptase-polymerase chain reaction analysis for human OCT4, NANOG, GDC, and GAPDH in A549 cancer and clone cells. Values shown below the gel are fold change for the respective genes in clone cells compared to cancer cells. The GAPDH values were normalized

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Fourier transform infrared studies of A549 cancer and clone cells

Studies were performed in A549 cancer and clone cells to understand the differences in their spectral properties by FT-IR spectroscopy [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h, [Figure 4]i; [Supplementary Figure S1]a, [Supplementary Figure S1]b, [Supplementary Figure S1]c, [Supplementary Figure S1]d, [Supplementary Figure S1]e, [Supplementary Figure S1]f, [Supplementary Figure S1]g, [Supplementary Figure S1]h. IR spectra showed peaks corresponding to amide I and amide II [Figure 4]i. Some background noise was observed in the spectra; however, it did not affect the pattern of the second derivative spectrum with/without subtracting the background.
Figure 4: Second derivative Fourier transform infrared spectra of A549 cancer and clone cells. Mean ± standard deviation values of three experiments were represented for A549 cancer (square) and clones (circle) cells. Wave numbers specific for; (a) A-DNA conformations/DNA-RNA hybrids at 970 and 1245 cm−1, (b) Z-DNA conformation at 1044 and 1067 cm−1, (c) C-O bond of carbohydrate at 1025 cm−1, (d and e) imidazole-metal interactions at 1088 and 1560 cm−1, (f) secondary structure of proteins at 1640, 1654, 1679 and 1696 cm−1, (g and h) lipid at 1390, 1450, and 1390 cm−1, (i) representative absorption spectrum of A549 clones and cancer cells of one experiment. *Significantly different at P< 0.05, **significantly different at P< 0.005. AU: Arbitrary unit

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Compared to differentiated cells, stem cells are known to have a higher ratio of nucleus to cytoplasm areas, a marker of changes in DNA compactness/conformation. Hence, in FT-IR studies, we have chosen regions (900–1250 cm −1) corresponding to nucleic acids.[29] At wave numbers 1044 and 1067 cm1, higher absorption was observed in A549 clones contributing from C-O stretching vibrations of DNA backbone specific to Z-form of DNA, especially at 1067 cm −1 (P < 0.005) [Figure 4]b; [Supplementary Figure S1]b. However, the peak intensity at 1215 cm −1, the another IR spectral marker of Z-form of DNA,[29] did not show a significant difference. The difference in our observation compared to the previous study might be due to use of whole cells in our study. FT-IR absorption at 970 cm −1 arising from deoxyribose main chain vibrations (C4'C5'H and O5'C5'H) of A-form of DNA was significantly lower in A549 clones compared to cancer cells [Figure 4]a; [Supplementary Figure S1]a. There was higher peak intensity at 1245 cm −1 corresponding to anti-symmetric PO2 vibration in cancer cells; however, it was not significant.

In our study, spectra at 1025 cm −1 representing for symmetric vibrations of C-O bond of carbohydrates was significantly higher (P < 0.005) in A549 clones as compared to cancer cells [Figure 4]c; [Supplementary Figure S1]c, which may arise from higher glycoprotein content in clones compared to cancer cells.

The spectra analyzed for IR markers of imidazole-metal interactions showed higher absorption at 1088 (corresponding to histidine C5Nτ metal complexes) and 1560 cm −1 (corresponding to υ [C4C5] histidine ligands of copper) in clones compared to cancer cells [Figure 4]d and [Figure 4]e; [Supplementary Figure S1]d and [Supplementary Figure S1]e. Previously, the increased intensities at these wave numbers were shown to be specific for metalloproteins.[27],[30]

Moreover, we have analyzed FT-IR spectra of A549 cancer and clone cells in the region of 1620–1710 cm −1 corresponding to the secondary structure of proteins [Figure 4]f; [Supplementary Figure S1]f. The peak intensity at wave number 1640 cm −1 corresponding to beta-sheet structure of proteins was significantly higher in A549 cancer cells as compared to clones. The other two peaks at wave numbers 1679 and 1696 cm −1 also representing beta proteins were not significantly different in A549 cancer and clone cells. In contrary, the peak intensity at wave number 1654 cm −1 representing the α-helix proteins was significantly higher in A549 clones compared to cancer cells. Furthermore, absorption associated with lipid regions at 1740, 1450, and 1390 cm −1 was shown to be significantly higher in clones compared to cancer cells [Figure 4]g and [Figure 4]h; [Supplementary Figure S1]g and [Supplementary Figure S1]h.

Circular dichroism spectroscopy studies of A549 cancer and clone cells

Our FT-IR studies showed overall alteration in the content/conformation of proteins and nucleic acids in A549 clones compared to cancer cells. To further explore the nature of these alterations, CD spectroscopy of A549 clone and cancer cells was performed in whole cells in the far UV and near UV regions. As shown in [Figure 5], the CD signal from A549 clones and cancer cells in far-UV region showed negative peaks at 209 and 222 nm, a typical characteristic of alpha-helical proteins. Even though the same number of cells (3 × 105) were taken for A549 clones and the cancer cells, it was interesting to observe that A549 clones showed more negative CD signal in 200–250 nm wavelength range compared to the cancer cells suggesting higher level of alpha-helical proteins in A549 clones, which is consistent with the FT-IR observations [Figure 4]f; [Supplementary Figure S1]f. The CD signal in the near UV region (260–280 nm) was more positive for A549 clones compared to the cancer cells, which may be attributed to higher level of Z-form of DNA in clones also observed by FT-IR studies [Figure 4]b; [Supplementary Figure S1]b. The CD signal below 200 nm could not be recorded due to sharp increase in signal to 800–1000 V, which makes the measurement unreliable at these wavelengths.
Figure 5: Circular dichroism spectrum of clones (dotted line) and A549 cancer cells (continuous line)

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Activity of superoxide dismutase in A549 cancer and clone cells

In A549 clones, an increased imidazole-metal vibration was observed in FT-IR studies [Figure 4]d and [Figure 4]e; [Supplementary Figure S1]d and [Supplementary Figure S1]e. These alterations may be associated with higher activity of metalloproteins such as SOD corresponding to these wave numbers.[27],[28] To confirm this, SOD activity was measured in A549 cancer and clone cells. Our results showed ~2-folds higher SOD activity (P< 0.005) in A549 clones compared to the cancer cells [Figure 6]a.
Figure 6: (a) Superoxide dismutase activity (U/μg) in A549 cancer and clone cells. (b) Clonogenic survival of A549 cancer and clone cells after 2 and 6 Gy of gamma irradiation. *Significantly different at P< 0.05

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Radiosensitivity of A549 cancer and clone cells

Increased SOD activity observed in A549 clone cells might be associated with their radioresistance. Hence, clonogenic assay was performed in A549 cancer and clone cells after 2 and 6 Gy of g-radiation. Results showed higher (P < 0.02) survival fraction of g-irradiated A549 clones compared to irradiated A549 cancer cells [Figure 6]b suggesting radio-resistant nature of A549 clone cells.


 > Discussion Top


In the present study, clones enriched with CSCs in A549 cells were isolated using SCA, which were characterized by cell surface marker CD44+/CD24−/low, Hoechst dye efflux assay, and expression of stemness genes (OCT4, NANOG, and GDC). These clones were further studied for their spectral signatures using techniques such as FT-IR and CD spectroscopy. FT-IR study was previously used to characterize stem cells and CSCs.[16],[19],[20],[22] We have employed the SCA to obtain clones enriched with CSCs despite the limitations of tedious protocol and requirement of long-duration to obtain the clones. However, the technique is quite robust and characterizes the self-renewal ability of CSCs under physiological culture conditions. The stemness of the cells obtained from SCA was further validated with CD44+/CD24−/low, dye efflux methods and reverse transcriptase-PCR (RT-PCR) for stemness genes, which showed substantial enrichment of CSCs in cells obtained by SCA. Compared to absolute fraction of clones, only 80 and 66% cells showed markers of CSCs obtained from SCA in terms of CD44+/CD24−/low and Hoechst 33342−/low, respectively, which may be either due to population heterogeneity, a reported phenomenon in embryonic stem cell biology [35],[36] or may be due to methodology associated experimental variation. However, it was clearly evident by RT-PCR data that clones showed higher expression of stemness genes compared to corresponding cancer cells.

Our FT-IR studies showed higher absorption at peaks (970 and 1245 cm −1) for A549 cancer compared to clone cells suggesting higher A-form of DNA in cancer cells. The higher transcription in fast proliferating cancer cells may be associated with their higher content of A-form of DNA, which also represent DNA-RNA hybrid.[29] In the previous studies, effect of Mie scattering was studied on amide I band region at 1730 cm −1 using poly (methyl methacrylate) spheres (particle size 5–15 μm) which were nonabsorbent to IR. Moreover, in these studies, IR was focused on a single particle using appropriate narrow aperture.[37],[38],[39] Since in our study, IR light gets exposed to whole FT-IR window and mammalian cells absorb IR light, the contribution of Mie scattering will be insignificant. It is also to be noted that since cells are in μm size range, Rayleigh scattering will be insignificant in IR region.[40]

Even though Z-form of DNA was discovered in 1979, their cellular functions have not been well illustrated in the literature, especially in stem cell biology. Z-form of DNA is known for transient conformational changes during transcription for selected genes, which may play a critical role in dynamics of CSCs in tumor microenvironment. Z-form of DNA has been shown to control the expression of C-MYC gene, and this conformation gets quickly reverted to B-DNA when C-MYC transcription switched off.[41],[42]C-MYC is known to regulate embryonic, pluripotent genes such as OCT-4, SOX-2, and KLF-4.[43] Hence, the higher Z-form of DNA in A549 clone cells may be attributed to higher fraction of CSCs and also contributing in stemness of these cells.

CSCs are known to express a higher level of surface markers constituting glycoproteins,[44] which might explain the higher level of C-O bands of carbohydrates in A549 clone cells. Moreover, variation in carbohydrate content in cells located at different locations of human embryonic stem cell (hESC) colony has been shown by Raman spectroscopy.[45] However, the difference in carbohydrate content during stem cell differentiation is not clear.

Higher signal for imidazole metal interactions in clones enriched with CSCs observed in FT-IR studies was further corroborated with increased SOD activity (metalloprotein) and more survival fraction of irradiated A549 clone cells. SOD is one of the well-known cellular anti-oxidant enzymes, which along with other cellular defence systems, such as glutathione and catalase, scavenge the free radicals generated and thus prevents damage to biomolecules after radiation exposure. The observed radioresistance in A549 clone cells is in agreement with the previous report of radioresistance of glioma CSCs [7] and increased SOD activity in A549 clone cells may be attributed to their lower radiosensitivity. These observations may have the clinical implication to radioresistance in lung cancer patients during radiotherapy.

The IR absorption for lipid band regions is more in A549 clones compared to cancer cells, which was in agreement with the literature showing higher lipid as marker for embryonic stem cells.[31] Our results did not show difference between A549 clone and cancer cells at 2920 cm −1 as reported previously for lipid band [31] in hESCs and the difference in our observation may be due to different cell systems used. hESCs are known to exhibit changes in the secondary structure of cellular proteins and similar feature were exhibited in FT-IR studies of A549 clone cells. The higher level of proteins with beta-sheet structure in A549 cancer cells could be explained by their fast proliferating nature. On the contrary, the higher level of proteins with alpha-helical content in A549 clone cells may arise from higher level of gap junction or other alpha-helical proteins. Previous study showed increase in secondary structure of proteins in murine embryonic stem cells.[16] Besides, our study distinguishes the beta and alpha structure of proteins in A549 clones enriched with CSCs. The increase in proteins with alpha-helical content in A549 clones were further confirmed by CD spectroscopy and validated by higher expression of major gap junction protein, connexin-43 in A549 clone cells [Supplementary Figure S2].




 > Conclusion Top


Our study used CD spectroscopy for characterizing live clones enriched with CSCs and also showed novel imidazole metal interactions by FT-IR studies, which were further, corroborated by activity of cellular SOD and increased radio-resistance. However, these findings need to be further extended using experimental animal models and to evaluate the fraction of CSCs in patient samples to make the knowledge more relevant for CSC based prognosis of cancer recurrence and therapy.

Acknowledgments

Dr. Murali M. S. Balla would like to acknowledge the K. S. Krishnan Fellowship from Department of Atomic Energy, Government of India. Authors would like to acknowledge the technical assistance of Mrs. Vasumathy Pillai, Mr. Manjoor Ali during confocal microscopy and Shri Sanjay Shinde for irradiation using Bhabhatron-II.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

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    Tables

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