Probing micro-environment of lipid droplets in a live breast cell: MCF7 and MCF10A

Chemical Physics Letters 670 (2017) 27–31

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Research paper

Probing micro-environment of lipid droplets in a live breast cell: MCF7 and MCF10A Catherine Ghosh a, Somen Nandi a, Kankan Bhattacharyya a,b,⇑ a b

Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal 462066, Madhya Pradesh, India

a r t i c l e

i n f o

Article history: Received 23 November 2016 In final form 29 December 2016 Available online 31 December 2016 Keywords: Breast cancer cells Lipid droplets (LDs) Polarity Solvation dynamics

a b s t r a c t Local environment of the lipid droplets inside the breast cancer cells, MCF7 and in non-malignant breast cells, MCF10A is monitored using time-resolved confocal microscopy. For this study, a coumarin-based dye C153 has been used. The local polarity and the solvation dynamics indicate that a cytoplasmic lipid droplet is less polar and displays slower solvation dynamics compared to the cytosol. Significant differences in terms of number of lipid droplets, polarity and solvation dynamics are observed between the cancer cell (MCF7) and its non-malignant cell (MCF10A). Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction Lipid droplets (LDs) play an important role in cellular lipid homeostasis and energy metabolism. They contain neutral lipid molecules like triacylglycerols, cholesteryl esters or retinyl esters surrounded by a phospholipid monolayer and proteins at the surface [1]. Lipid droplets store triglycerides for energy. They also store phospholipids and sterols, which are fundamental for the growth and maintenance of the cell membrane and in cellular metabolism [2]. They are implicated in the development of metabolic and infectious diseases such as atherosclerosis, liver damage, lipodystrophy insulin resistance, and Chanarin-Dorfman syndrome [3]. Lipid droplets have significant roles in many aspects of cancer [3–9]. It is becoming increasingly evident that lipid droplets are important biomarkers of cancer [8,10–12]. Cancer cells are known to contain increased numbers of Lipid droplets compared with normal cells. This has been observed in colon adenocarcinomas [8]. The lipid droplet-rich cancer cells are more resistant to chemotherapy [13]. Raman-based imaging is being used to define lipid droplets in tumors. This is an emerging tool for monitoring or predicting drug treatment response in cancer [14,15]. Abramczyk et al. have assessed the impact of cancer aggressiveness on the amount of cytosolic lipid droplets in non-malignant and malignant human breast epithelial cell-lines. They have also analyzed the composition of lipid droplets in non-malignant and malignant ⇑ Corresponding author at: Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal 462066, Madhya Pradesh, India. E-mail address: [email protected] (K. Bhattacharyya). http://dx.doi.org/10.1016/j.cplett.2016.12.068 0009-2614/Ó 2016 Elsevier B.V. All rights reserved.

human breast epithelial cell-lines [1]. Lipid droplets have also been extensively studied by vibrational spectroscopy [16–19]. Lipid droplets have been studied previously using various fluorescent probes [1,20,21]. In this work, we attempt to study the local environment of lipid droplets inside live breast cancer cells (MCF7) and non-malignant breast cells (MCF10A) using a fluorescent probe coumarin 153 (C153, Scheme 1). Recently, we have studied and compared the micro-environment of a cancer cells to its non-cancer counterpart in various way such as by polarity, solvation dynamics, fluorescence intensity oscillations etc [22–26]. In this present work, we have studied the polarity and solvent microenvironment properties inside the lipid droplets as well as in the cytosol of a live breast cancer cell (MCF7) and a non-cancer cell (MCF10A) using time-resolved confocal microscopy. Subsequently, a comparison between cancer cells and non-cancer cells has also been made. For this study, C153 dye has been used to stain both the cell-lines.

2. Experimental section 2.1. Materials Laser-grade dye, C153 (Scheme 1) from Exciton Inc and Dimethylsulfoxide (DMSO) for cell culture from Sigma Aldrich were used as received. Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Sigma Aldrich), Trypsin-EDTA solutions (Sigma Aldrich), Pen Strep Glutamine (Gibco) and MEGM Bullet kit (Lonza) were obtained from the respective companies. Penicillin-

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C. Ghosh et al. / Chemical Physics Letters 670 (2017) 27–31

O

O

Shamrock series), attached to one of the external port of our confocal microscope was used to record the emission spectra [22,25]. To avoid the photo-bleaching of the live cells, very low laser power (
10 nW) has been applied and also we allowed small data acquisition time while recording emission spectra.

N

CF3 Scheme 1. Structure of coumarin 153 (C153).

Streptomycin and Fetal Bovine Serum (FBS) were purchased from Invitrogen. All chemicals were used without further purification. 2.2. Methods 2.2.1. Cell culture Detailed procedures for the preparation of cell-lines were discussed in our previous publications [24,25,27,28]. Human breast cancer cell, MCF7 was grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% Pen Strep Glutamine in an atmosphere of 5% CO2 enriched air at 37 °C. The human breast epithelial cell-line, MCF10A was received from the American Type Culture Collection (Manassas, VA) and grown in Lonza formulated medium, MEGM supplemented with antibiotics (100 units/ml Penicillin and 100 lg/ml Streptomycin) in 5% CO2 incubator at 37 °C. The cells were then seeded on 30 mm confocal petri dish for 24 h. Before the staining of the cell-lines, a stock solution of C153 in biocompatible DMSO was prepared. 200 lL of 500 nM dye solutions was added to the culture dish and incubated for 4 h. Prior to use for experiments, the cells were washed 3–4 times with phosphate buffered saline (PBS) to remove trace amount of dye outside the cell surface and 200 lL fresh phenol red free media was added. All the experiments were executed at 20 °C. To prevent photo-damage of the live cells, we have recorded confocal images at very low laser power (
200 nW). 2.2.2. Time resolved confocal microscopy The set up for our confocal microscope (PicoQuant, MicroTime 200) is described in details in our previous publication [22,24,25]. A dichroic mirror (405DCXR, Chroma) and appropriate filter (HQ430lp, Chroma) were used to separate the fluorescence from the excitation laser (PDL 828-S ‘SEPIA II’) at 405 nm. The fluorescence with different polarizations (parallel, Ik and perpendicular, I\) was separated using a polarizer cube (Chroma). The signals were detected with two separate detectors (micro photon device, MPD). TCSPC decays were recorded at specific emission wavelengths using appropriate narrow band-pass filters (e.g. XBPA480, 510 etc., Asahi Spectra). Ik and I\ components of the fluorescence were combined to generate the fluorescence decay at magic angle conditions, which is written as follows [22,25] 2

Imagic ðtÞ ¼ Ik ðtÞ cos2 ð54:750 Þ þ I? ðtÞG sin ð54:750 Þ ¼ ð1=3ÞIk ðtÞ þ ð2=3ÞGI? ðtÞ

ð1Þ

G factor was measured as described earlier [22] and was found to be
1.7. Fluorescence decays have been deconvoluted using the Instrument Response Function (IRF) and analyzed by DAS6 v6.3 software. IRF was recorded by back-scattering the light from a bare slide using a laser diode emitting at 405 nm. The FWHM (full width at half maximum) of the IRF is
100 ps. 2.2.3. Fluorescence spectra under a confocal microscope An electron multiplying charge-coupled device (EMCCD, ANDOR Technology) and a spectrograph (ANDOR Technology,

2.2.4. Analysis of solvation dynamics The time-resolved emission spectra (TRES) of C153 inside of the different regions of live cell were constructed from the steady-state emission spectra and the picosecond fluorescence transients recorded under the microscope as described by Maroncelli and Fleming [29]. The instrument response function (IRF) of our picosecond setup, as already mentioned earlier, is
100 ps. Therefore, the ultrafast component of solvation dynamics occurring in a time scale shorter than 100 ps would be certainly missed. This percentage of solvent missed has been calculated by following the procedure given by Fee and Maroncelli [30]. They proposed a simple method for the calculation of true emission frequency at time zero i.e. mtheo ð0Þ. In order to detect the percentage missed of solvation, we have used Fee-Maroncelli equation as follows


 np Vtheo ð0Þ ¼ vsystem  vnp abs abs  vem

ð2Þ

where m ð0Þ represent theoretical emission maximum at zero and mnp time. mnp em denote absorption and emission maxima of abs C153 in a non-polar solvent (cyclohexane/or n-hexane), respecindicates the absorption maximum of the system. tively. msystem abs theo

msystem could not be recorded under a few molecules condition i.e. abs under confocal microscope. Thus, we assumed that msystem is equal abs

to the absorption maximum of C153 in a solvent where C153 exhibit similar emission maximum as that in the live cell. Using this approximation, percentage missed has been calculated. Solvation dynamics is described by the decay of the solvent correlation function C(t), defined as

CðtÞ ¼

v ðtÞ  v ð1Þ v ð0Þ  v ð1Þ

ð3Þ

where m(0), m(t), and m(1) are the emission maxima (frequencies) at times 0, t, and 1, respectively. The total dynamic Stokes shift (DSS, Dm) is given by the denominator on the right-hand side of Eq. (3). The solvent correlation functions (C(t)) were fitted to a single- or double-exponential decay as follows

CðtÞ ¼

X

ai et=si

ð4Þ

3. Results and discussion 3.1. Confocal images of MCF7 and MCF10A stained by C153 Fig. 1A and B shows the confocal images of MCF7 and MCF10A cells stained by C153 dye, respectively. It is easily seen that C153 stains the cytosol of both the breast cancer cell (MCF7) and normal breast cell (MCF10A). In addition to the cytosol, C153 also stains a large number of spherical dots in the cancer cell. Such bright spherical dots are fewer in number in the non-cancer cell. Many groups previously assigned these spherical bright dots to lipid droplets [31–34]. Recently, we have also reported that C153 specifically stains lipid droplets [22,23,35]. Following previous work, these bright spherical dots stained by C153 in the cytosol of both the cell lines are identified as lipid droplets. It is quite obvious from the confocal images that the number of lipid droplets in MCF7 (cancer cell) is higher than in MCF10A (non cancer cell). The higher prevalence of lipid droplets in cancer cells as compared to non-cancer cells has also been reported previously [22,24]. The reasons for this may be accounted as follows. In cancer

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C. Ghosh et al. / Chemical Physics Letters 670 (2017) 27–31

MCF7

(A)

MCF10A

(B)

Fig. 1. Confocal images: (A) MCF7 and (B) MCF10A, stained by C153 dye.

cells the rates of glycolysis, lactate production and biosynthesis of lipids, nucleic acids and other bio-macromolecules are very high [36]. This enhanced rate of glycolysis is needed to produce adequate energy during rapid cell division in cancer cells. In the absence of sufficient oxygen, glucose is partially oxidized to pyruvate instead of CO2 and H2O. During glycolysis some of the pyruvate formed gets converted into citrate. Some of the mitochondrial citrate gets spread over the cytosol and produces fatty acids and cholesterol which are the precursors of lipid droplets. This leads to increased number of lipid droplets in cancer cells. This is known as Warburg effect [37]. 3.2. Emission maxima and polarity of cytosol and lipid droplets Fig. 2 shows the emission spectra of C153 inside MCF7 and MCF10A cells recorded under the confocal microscope. The values of emission maxima of C153 and the corresponding polarity at the lipid droplets and cytosol of both the cell lines are given in Table 1. Inside the cytosol of MCF7, C153 exhibits emission maximum (kem max ) at
534 nm. This corresponds to dielectric constant, e
26 (between that of ethanol and methanol [29]). From the value of kem max of C153 inside the cytosol of MCF10A (
527 nm), the e is estimated to be same as that of n-propanol (e
24) [29]. Unlike lipid droplets, the cytosol in MCF7 is more polar than that of MCF10A. Also the polarity gradient between lipid droplets and cytosol is greater for MCF7 than for MCF10A.

1.0

Cyto (MCF10A) Cyto (MCF7) LD (MCF10A) LD (MCF7)

Fluorescence Intensity

0.8

0.6

0.4

0.2

0.0 450

500

550

600

Wavelength (nm) Fig. 2. Emission spectra of C153 inside MCF7 and MCF10A cell.

The emission maximum (kem max ) of C153 in the lipid droplets of cancer cell, MCF7 (
491 nm) is blue shifted from that of noncancer cell, MCF10A (
495 nm). Thus the polarity of lipid droplets in the cancer cell is lower than that of non-cancer cell. The kem max
491 nm of C153 in lipid droplets of MCF7 is very close to that of C153 in n-butyl acetate (490 nm) [29]. Therefore, the polarity (e) of lipid droplets in MCF7 is almost same as that of n-butyl acetate (e
4.0). The kem max of C153 in lipid droplets of MCF10A (
495 nm) is in between that of C153 in n-butyl acetate (490 nm) and ethyl acetate (501 nm). As a result the polarity of the lipid droplets is in between that of n-butyl acetate (e
5.0) and ethyl acetate (e
6.0) [38], i.e. e of lipid droplets in noncancer cells is
5.5. For both the cell-lines we observed that the emission maxima (kem max ) of C153 is blue shifted in the lipid droplets compared to that in cytosol indicating lower polarity of the lipid droplets. This trend has been observed previously for both cancer and non-cancer cells [22,23,35]. 3.3. Solvation dynamics in cytosol and lipid droplets of MCF7 and MCF10A From the previous section, we have seen that polarity gradient is greater for cancer cell, MCF7 than for the non-cancer cell, MCF10A. This is likely to affect polar reactions (reactions where transition states are more polar than the reactants) occurring inside the cells. Polar reactions are hindered due to ultraslow component of solvation of biological water (i.e. water present in biological systems). It may be noted here that in bulk water solvation exhibits a component of 1 ps time scale [39–41]. However in many nano-confined media/or in biological assembly, water displays a component of
100–1000 ps [42–44]. To understand how polar reactions are influenced inside the cells, we carried out solvation dynamics experiment. Fig. 3 shows the fluorescent transients of C153 recorded inside lipid droplets and cytosol of both MCF7 and MCF10A cells. For all the transients recorded at long emission wavelengths, there is a distinct rise component. Thus, decay at short emission wavelength i.e. blue end and rise preceding the decay at long emission wavelength i.e. red end clearly illustrate that solvation dynamics is occurring inside both the cells. The emission spectra of lipid droplets and cytosol inside both cancer cell and non-cancer cell exhibit a gradual red shift with increase in time. The total dynamic Stokes Shift (DSS) and emission frequencies at time zero, m0, of all the systems have been calculated from the time resolved emission spectra (TRES, shown as insets in Fig. 3) and are given in Table 1. Fig. 4 shows the decay of the solvent correlation function,

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C. Ghosh et al. / Chemical Physics Letters 670 (2017) 27–31

Table 1 Emission maxima, dielectric constant and decay parameters of C(t) of C153 for the lipid droplets and cytosol in a breast cancer (MCF7) cell and in a non-cancer (MCF10A) cell Cell line

Emission maxima kmax (nm)

Dielectric constant (e)

Dma [m(0)] (cm1)

m(0)Theo cm1 (% missed)

s1b (a1) (ps)

s2c (a2) (ps)

hssolid (ps)

MCF7

Cytosol LD

534 491

26 4.0

1090 [19,770] 600 [20,992]

20,285 (30) 21,130 (15)

700 (0.70) 600 (0.35)

– 3500 (0.50)

500 2000

MCF10A

Cytosol LD

527 495

24 5.5

860 [19,850] 570 [20,708]

20,229 (30) 20,982 (30)

1100 (0.70) 1900 (0.70)

– –

800 1300

LD (MCF7)

Intensity (a.u.)

0.8

1.0 0.8 0.6

t

6000 ps 1400 ps 400 ps 0 ps

(B)

Cyto (MCF7)

0.4 0.2 0.0

0.6 0.4 0.2

IRF

1.0 Intensity (a.u.)

(A)

1.0

Intensity (a.u.)

hssoli = a1s1 + a2s2, ±100 ps where s1 and s2 are the individual components with corresponding amplitudes of a1 and a2, respectively. a ±100 cm1. b ±50 ps. c ±100 ps. d Average solvation time.

0.8 0.6 0.4 0.2 0.0

18000 21000 -1 Wavenumber (cm )

t

2200 ps 500 ps 0 ps

16000 20000 -1 Wavenumber (cm )

600 nm

600 nm

450 nm

450 nm

0.0 2

4

6

8

0

2

Time (ns) (C)

LD (MCF10A)

Intensity (a.u.)

0.8

1.0 Intensity (a.u.)

1.0

0.8 0.6

0.4 0.2

2

4

6500 ps 2000 ps 800 ps 0 ps

t

(D)

Cyto (MCF10A)

0.2

0.6

0

6

6

8

Time (ns)

0.4 0.0

0.0

4

1.0 Intensity (a.u.)

0

0.8 0.6

t

0.4 0.2 0.0

18000 21000 -1 Wavenumber (cm )

3500 ps 1000 ps 400 ps 0 ps

16000 20000 -1 Wavenumber (cm )

600 nm

600 nm

450 nm

450 nm

8

0

2

4

6

8

Time (ns)

Time (ns)

Fig. 3. Picosecond decays of C153 inside the LDs and cytosol: Upper panel for MCF7 cell and lower panel for MCF10A cell. Corresponding TRES are shown in insets. (kex = 405 nm).

1.0

MCF7

1.0

(A)

(B)

0.8

0.8

Cytosol LD

0.6 0.4

Cytosol LD

0.6

C(t)

C(t)

MCF10A

0.4 0.2

0.2 0.0

0.0 0

1500

3000

Time (ps)

4500

6000

0

1500

3000

4500

Time (ps)

6000

Fig. 4. Decay of C(t) inside: (A) MCF7, (B) MCF10A cell. Data points denote values of C(t) and solid lines indicate the best fit.

C. Ghosh et al. / Chemical Physics Letters 670 (2017) 27–31

C(t). We have fitted the C(t) in single/or bi-exponential decay to obtain the best fit. The fitting parameters of the decay have been listed in Table 1. The lipid droplets of breast cancer cell (MCF7) exhibit two components of solvation dynamics:
600 ps (35%) and
3500 ps (50%) and 15% ultrafast component which is missed in our set up with an average solvation time (hssoli) of
2000 ps. On the other hand the cytosol exhibits single component decay with the average solvation time of
500 ps. Thus the average solvation time in lipid droplets is
4 times slower than in cytosol. This slow time scale in solvation dynamics indicates that the lipid environment in the lipid droplets is highly restricted [28–31] and unfavoured the polar reactions. In the case of non-cancer (MCF10A) cells, the decay of C(t) of C153 in lipid droplets and cytosol is found to be single exponential (time constants are
1900 ps (70%) and
1100 ps (70%) respectively). Considering that 30% of the solvation is missed in our set up, the time constants (ssol) are obtained of
1300 ps and
800 ps, respectively. It is evident that the time scale of solvation dynamics in lipid droplets of non-cancer (MCF10A) cells is
1.5 times faster compared to that in cancer (MCF7) cells. This illustrates that the lipid droplets inside the non-cancer cells favors the polar reaction to some extent compared to the cancer cells. In contrary, the cytosol of MCF10A cells display
1.5 times slower solvation dynamics compared to the MCF7 cells. Slower solvation dynamics in lipid droplets are likely to affect polar reactions by slowing them down. The large number of lipid droplets present inside the breast cancer cell provides a hydrophobic environment where the hydrophobic species such as cell signaling agents may accumulate. Confinement of non-polar cell signaling agents inside the lipid droplets might favor non-polar reactions and inhibit polar reactions. It may be recalled here that the ability of the water molecules to re-organize in response to an applied electric field dictates the polarity or dielectric constant of the medium [45]. The individual dipole moment of one water molecule is increased due to hydrogen bonding between water molecules which results in mutual dynamic polarization. This leads to an increase in the dielectric constant of the medium [46]. In the lipid droplets, water-water hydrogen bonding is lost to a large extent and most of the water molecules get partially immobilized by binding to the macromolecules. This reduces the contribution of dynamic polarization and the result eventually lead to the lowering of polarity and slower solvation dynamics in the lipid droplets compared to those in the cytosol. 4. Conclusion In this work, we have investigated the differences in polarity and time scales of solvation dynamics in lipid droplets and cytosol of a live breast cell (MCF10A) and a breast cancer cell (MCF7) using time and space-resolved confocal microscopy. It is found that the polarity in lipid droplets is lower than that in the cytosol for both the cell-lines. The time scale of solvation dynamics inside the lipid droplets is found to be slower than that in cytosol. We demonstrate that there are significant differences observed in terms of number of lipid droplets, polarity and solvation dynamics between the breast cancer cells and non-cancer cells. The plausible reasons and implications have been discussed thoroughly. These results may provide new insights into the abnormal biochemistry of cancer cells.

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