Madalina Andreea Badea1*, Mihaela Balas1, Florentina Gina ...
1
Madalina Andreea Badea 1* , Mihaela Balas 1 , Florentina Gina Cojocaru 2 , Daniela Ionita 3 , Anca Dinischiotu 1 1 University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, 91-95 Splaiul Independentei, R-050095, Bucharest, Romania. 2 University of Bucharest, Faculty of Biology, Department of Anatomy, Physiology and Biophysics, 91-95 Splaiul Independentei, R-050095, Bucharest, Romania. 3 Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of General Chemistry, 313 Splaiul Independentei, 060042, Bucharest, Romania. *Corresponding author: [email protected] Background: Triple negative breast cancer has a phenotype characterized by the absence of progesterone and estrogen receptors and lack of HER2 overexpression (1). In order to find new strategies for treatment, single-walled carbon nanotubes (SWCNT) in combination with chemotherapeutics were studied and tested as new therapeutic tools (2). Aim: The objective of this study was to evaluate the efficiency of SWCNT in the transport of cisplatin (CDDP) for improving its cytotoxic effects on MDA-MB-231 cells. The nanoconjugates SWCNT-COOH-CDDP were obtained by functionalization of SWCNT with carboxyl groups (SWCNT-COOH) and conjugation with CDDP. MDA-MB-231 cell line (triple negative breast cancer cells) was cultured in Dulbecco′s Modified Eagle′s Medium. MDA-MB-231 cells were exposed to different doses of SWCNT-COOH, SWCNT-COOH-CDDP (0.01 – 2 μg/mL) and CDDP (0.00632 – 1.26 μg/mL) for 24 and 48 h. Spectrophotometric and fluorescence methods were performed for the evaluation of cellular viability (MTT test), reduced glutathione content (GSH) and determination of reactive oxygen species (ROS) production. Immunoblotting was performed for the assessment of Nrf2, caspase-3, caspase-8 and Bid proteins expressions. Wound healing assay was performed for the evaluation of the effects of SWCNT-COOH-CDDP on cell migration. Control CDDP SWCNT-COOH SWCNT-COOH-CDDP A. * * ** *** *** 0 20 40 60 80 100 120 140 Cellular viability (% of control) 24 h control 0.01 μg/mL SWCNT/0.0632 μg/mL CDDP 0.05 μg/mL SWCNT/0.0316 μg/mL CDDP 0.1 μg/mL SWCNT/0.632 μg/mL CDDP 0.25 μg/mL SWCNT/0.158 μg/mL CDDP 0.5 μg/mL SWCNT/0.316 μg/mL CDDP 1 μg/mL SWCNT/0.63 μg/mL CDDP 2 μg/mL SWCNT/1.26 μg/mL CDDP Cellular viability Control CDDP SWCNT-COOH SWCNT-COOH-CDDP B. * *** *** *** *** *** *** 0 20 40 60 80 100 120 Cellular viability (% of control) 48 h control 0.01 μg/mL SWCNT/0.0632 μg/mL CDDP 0.05 μg/mL SWCNT/0.0316 μg/mL CDDP 0.1 μg/mL SWCNT/0.632 μg/mL CDDP 0.25 μg/mL SWCNT/0.158 μg/mL CDDP 0.5 μg/mL SWCNT/0.316 μg/mL CDDP 1 μg/mL SWCNT/0.63 μg/mL CDDP 2 μg/mL SWCNT/1.26 μg/mL CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h 48 h * *** * * ** 0 20 40 60 80 100 120 140 160 180 Relative level of reduced glutathione content (% of control) GSH control 0.5 μg/mL SWCNT/0.316 μg/mL CDDP 1 μg/mL SWCNT/0.632 μg/mL CDDP Oxidative stress * * *** *** ** * * * *** * 0 20 40 60 80 100 120 140 Relative protein expression of Nrf2 (% of control) Nrf2 control 0.5 μg/mL SWCNT/0.316 μg/mL CDDP 1 μg/mL SWCNT/0.632 μg/mL CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h 48 h (b) C CDDP SWCNT- COOH SWCNT- COOH-CDDP 0 0.316 0.632 0.5 0.5 1 1 μg/mL 100 kDa 43 kDa Nrf2 β-actin 24 h C CDDP SWCNT- COOH SWCNT- COOH-CDDP 0 0.316 0.632 0.5 0.5 1 1 μg/mL 100 kDa 43 kDa Nrf2 48 h β-actin (a) Cell death C SWCNT -COOH SWCNT- COOH-CDDP CDDP 48 h C SWCNT -COOH SWCNT- COOH-CDDP CDDP Cleaved caspase-8 (p18) Procaspase-8 (p54/55) Cleaved caspase-8 (p43/41) β-actin ** ** *** ** ** 0 20 40 60 80 100 120 140 160 180 Relative protein expression of procaspase-8/caspase-8 (% of control) Procaspase-8/Caspase-8 Procaspase-8 Caspase-8 Control CDDP SWCNT- COOH SWCNT- COOH-CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h 48 h μg/mL 0 0 0.632 1 1 0.632 1 1 0 0.632 1 1 0 0.632 1 1 μg/mL 54/55 kDa 18 kDa 24 h 43/41 kDa (a) 43 kDa (b) 24 h 48 h Cell migration Control 0.316 μg/mL CDDP 0.632 μg/mL CDDP 0.5 μg/mL SWCNT-COOH 1 μg/mL SWCNT-COOH 0.5 μg/mL SWCNT- COOH-CDDP 1 μg/mL SWCNT- COOH-CDDP ** *** *** 0 20 40 60 80 100 120 Wounding healing (fold of time point 0 h) Cell migration 24 h 48 h Control CDDP SWCNT-COOH SWCNT-COOH-CDDP 0 0.316 0.632 0.5 1 0.5 1 μg/mL o The level of GSH raised after 24 and 48 h of exposure to 0.5 μg/mL SWCNT-COOH-CDDP, while a decrease until 78.31% was recorded after 48 h in the presence of 1 μg/mL nanoconjugates. o The expression of Nrf2 decreased until 33% after 24 h of treatment with 1 μg/mL SWCNT-COOH-CDDP and increased until 80% compared to control (100%) after 48 h. * * * * * * 0 1000 2000 3000 4000 5000 6000 RFU (ex. 485 nm/em. 520 nm) ROS control 0.5 μg/mL SWCNT/0.316 μg/mL CDDP 1 μg/mL SWCNT/0.632 μg/mL CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h 48 h o ROS level increased in a time and dose-dependent manner in the presence of nanoconjugates relative to control. o A slight increase was registered also for 1 μg/mL SWCNT-COOH after 24 and 48 h of incubation. o The cellular viability decreased in a time and dose-dependent manner in the presence of nanoconjugates relative to control. A. B. C. A. B. Cell viability after (A) 24 and (B) 48 h of exposure to SWCNT- COOH, SWCNT-COOH-CDDP (0.01 – 2 μg/mL) and CDDP (0.00632 – 1.26 μg/mL). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. Conclusions: these nanoconjugates induced apoptosis in MDA-MB-231 cells, probably by both intrinsic and extrinsic pathways, by triggering the oxidative stress mechanisms, and inhibited their migration potential. * * * * ** *** 0 500 1000 1500 2000 2500 Procaspase-3/Caspase-3 Procaspase-3 Caspase-3 * * * * 0 50 100 150 200 250 0 0.316 0.632 0.5 1 0.5 1 0 0.316 0.632 0.5 1 0.5 1 Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 48 h Relative protein expression of procaspase-3/caspase-3 (% of control) μg/mL A. Relative protein expression of (b) procaspase-3/caspase-3 and (c) Bid proteins after 24 and 48 h of exposure to 0.5, 1 μg/mL SWCNT-COOH and SWCNT-COOH- CDDP, 0.316, 0.632 μg/mL CDDP, respectively. In figure (b), the lower graph presents a magnified image of the scale range between 0–250 from the upper graph. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. μg/mL 32 kDa 43 kDa 24 h 20/17/11 kDa μg/mL 32 kDa 43 kDa 48 h 20/17/11 kDa (a) 0 0.316 0.632 0.5 0.5 1 1 0 0.316 0.632 0.5 0.5 1 1 C CDDP SWCNT- COOH SWCNT- COOH-CDDP C CDDP SWCNT- COOH SWCNT- COOH-CDDP Procaspase-3 β-actin Cleaved caspase-3 Procaspase-3 β-actin Cleaved caspase-3 (b) Cell migration (scratch wound healing assay). (A) Bright-field images presenting the MDA-MB-231 cell culture after 24 and 48 h of wounding and incubation with 0.5, 1 μg/mL SWCNT-COOH and SWCNT-COOH-CDDP, 0.316, 0.632 μg/mL CDDP, respectively. (B) Quantification of presented images. o The inhibition of the cell migration was observed after 24 and 48 h of exposure with 1 μg/mL SWCNT-COOH-CDDP. o A dose of 0.632 μg/mL increased the migration capacity of MDA-MB-231 after 24 h of treatment. *** *** *** ** ** * ** ** *** 0 20 40 60 80 100 120 140 160 180 200 Relative protein expression of Bid (% of control) Bid control 0.5 μg/mL SWCNT - 0.316 μg/mL CDDP 1 μg/mL SWCNT - 0.632 μg/mL CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP Control CDDP SWCNT- COOH SWCNT- COOH-CDDP 24 h 48 h 22 kDa Bid 22 kDa Bid (c) Keywords : breast cancer; carbon nanotubes; cisplatin; apoptosis B. Relative protein expression of procaspase-8/caspase-8 after 24 and 48 h of exposure to 0.5, 1 μg/mL SWCNT- COOH and SWCNT-COOH-CDDP, 0.316, 0.632 μg/mL CDDP, respectively. ** p < 0.01, *** p < 0.001 vs. control. o Caspase-3 and caspase-8 were activated in the presence of 1 μg/mL SWCNT-COOH-CDDP starting with 24 h of treatment. o An increase of procaspase- 3 protein expression was observed after the treatment with SWCNT- COOH-CDDP. o The expression of Bid protein was upregulated after 24 h of incubation with nanoconjugates, followed by a downregulation at 48 h. A dose with 1 μg/mL SWCNT- COOH-CDDP induced downregulation of Bid protein expression at both intervals tested. References: (1) Collignon, J.; Lousberg, L.; Schroeder, H.; Jerusalem, G. Triple-negative breast cancer: treatment challenges and solutions. Breast Cancer (Dove Med. Press). 2016, 8:93-107. (2) Rastogi, V.; Yadav, P.; Bhattacharya, S.S.; Mishra, A.K.; Verma, A.; Pandit, J.K. Carbon nanotubes: An emerging drug carrier for targeting cancer cells. J. Drug Deliv. 2014, 670815. Acknowledgments to PN-III-P2-2.1-PED-2016-0904 project. A. B.
Transcript of Madalina Andreea Badea1*, Mihaela Balas1, Florentina Gina ...
Madalina Andreea Badea1*, Mihaela Balas1, Florentina Gina Cojocaru2, Daniela Ionita3, Anca Dinischiotu1
1University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, 91-95 Splaiul Independentei, R-050095, Bucharest, Romania.2University of Bucharest, Faculty of Biology, Department of Anatomy, Physiology and Biophysics, 91-95 Splaiul Independentei, R-050095, Bucharest, Romania.3Politehnica University of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of General Chemistry, 313 Splaiul Independentei, 060042,Bucharest, Romania.
*Corresponding author: [email protected]
Background: Triple negative breast cancer has a phenotype characterized by the absence of progesterone and estrogen receptors and lack of HER2 overexpression(1). In order to find new strategies for treatment, single-walled carbon nanotubes (SWCNT) in combination with chemotherapeutics were studied and tested as new
therapeutic tools (2). Aim: The objective of this study was to evaluate the efficiency of SWCNT in the
transport of cisplatin (CDDP) for improving its cytotoxic effects on MDA-MB-231 cells.
The nanoconjugates SWCNT-COOH-CDDP wereobtained by functionalization of SWCNT with carboxylgroups (SWCNT-COOH) and conjugation with CDDP.
MDA-MB-231 cell line (triple negative breast cancercells) was cultured in Dulbecco′s Modified Eagle′sMedium.
MDA-MB-231 cells were exposed to different doses of SWCNT-COOH, SWCNT-COOH-CDDP (0.01 – 2 µg/mL) and CDDP (0.00632 – 1.26 µg/mL) for 24 and 48 h.
Spectrophotometric and fluorescence methods wereperformed for the evaluation of cellular viability (MTTtest), reduced glutathione content (GSH) anddetermination of reactive oxygen species (ROS)production.
Immunoblotting was performed for the assessment of Nrf2, caspase-3, caspase-8 and Bid proteins expressions.
Wound healing assay was performed for the evaluation of the effects of SWCNT-COOH-CDDP on cell migration.
Control CDDP SWCNT-COOH SWCNT-COOH-CDDP
A.
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Ce
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24 h
control 0.01 µg/mL SWCNT/0.0632 µg/mL CDDP
0.05 µg/mL SWCNT/0.0316 µg/mL CDDP 0.1 µg/mL SWCNT/0.632 µg/mL CDDP
0.25 µg/mL SWCNT/0.158 µg/mL CDDP 0.5 µg/mL SWCNT/0.316 µg/mL CDDP
1 µg/mL SWCNT/0.63 µg/mL CDDP 2 µg/mL SWCNT/1.26 µg/mL CDDP
Cellular viability
Control CDDP SWCNT-COOH SWCNT-COOH-CDDP
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48 hcontrol 0.01 µg/mL SWCNT/0.0632 µg/mL CDDP
0.05 µg/mL SWCNT/0.0316 µg/mL CDDP 0.1 µg/mL SWCNT/0.632 µg/mL CDDP
0.25 µg/mL SWCNT/0.158 µg/mL CDDP 0.5 µg/mL SWCNT/0.316 µg/mL CDDP
1 µg/mL SWCNT/0.63 µg/mL CDDP 2 µg/mL SWCNT/1.26 µg/mL CDDP
Control CDDP SWCNT-
COOH
SWCNT-
COOH-CDDPControl CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
24 h 48 h
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GSH
control0.5 µg/mL SWCNT/0.316 µg/mL CDDP1 µg/mL SWCNT/0.632 µg/mL CDDP
Oxidative stress
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Re
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Nrf2
control0.5 µg/mL SWCNT/0.316 µg/mL CDDP1 µg/mL SWCNT/0.632 µg/mL CDDP
Control CDDP SWCNT-
COOH
SWCNT-
COOH-CDDPControl CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
24 h 48 h
(b)
C CDDP SWCNT-
COOHSWCNT-
COOH-CDDP
0 0.316 0.632 0.5 0.51 1µg/mL
100 kDa
43 kDa
Nrf2
β-actin
24 h
C CDDP SWCNT-
COOHSWCNT-
COOH-CDDP
0 0.316 0.632 0.5 0.51 1µg/mL
100 kDa
43 kDa
Nrf2
48 h
β-actin
(a)
Cell death
C SWCNT
-COOHSWCNT-
COOH-CDDPCDDP
48 h
C SWCNT
-COOH
SWCNT-
COOH-CDDPCDDP
Cleaved caspase-8 (p18)
Procaspase-8 (p54/55)
Cleaved caspase-8 (p43/41)
β-actin
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Procaspase-8/Caspase-8
Procaspase-8Caspase-8
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SWCNT-
COOH-CDDPControl CDDP SWCNT-
COOH
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COOH-CDDP
24 h 48 h
µg/mL 0 00.632 1 1 0.632 1 1
0 0.632 1 1 0 0.632 1 1µg/mL
54/55 kDa
18 kDa
24 h
43/41 kDa
(a)
43 kDa
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48 h
Cell migration
Control 0.316 µg/mL
CDDP0.632 µg/mL
CDDP
0.5 µg/mL
SWCNT-COOH
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SWCNT-COOH
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COOH-CDDP
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Cell migration24 h 48 h
Control CDDP SWCNT-COOH SWCNT-COOH-CDDP
0 0.316 0.632 0.5 1 0.5 1µg/mL
o The level of GSH raised after 24 and 48 h of exposure to 0.5 µg/mL
SWCNT-COOH-CDDP, while a decrease until 78.31% was recorded after 48 h in the
presence of 1 µg/mL nanoconjugates.
o The expression of Nrf2 decreased until 33% after 24 h of treatment
with 1 µg/mL SWCNT-COOH-CDDP and increased until 80% compared
to control (100%) after 48 h.
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0
1000
2000
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5000
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RF
U (
ex. 4
85
nm
/em
. 5
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0.5 µg/mL SWCNT/0.316 µg/mL CDDP
1 µg/mL SWCNT/0.632 µg/mL CDDP
Control CDDP SWCNT-
COOH
SWCNT-
COOH-CDDPControl CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
24 h 48 h
o ROS level increased in a time and dose-dependent manner in the
presence of nanoconjugates relative to control.
o A slight increase was registered also for 1 µg/mL SWCNT-COOH after 24
and 48 h of incubation.
o The cellular viability decreased in a time
and dose-dependent manner in the
presence of nanoconjugates
relative to control.
A.
B.
C.
A.
B.
Cell viability after (A) 24 and (B)48 h of exposure to SWCNT-
COOH, SWCNT-COOH-CDDP (0.01 – 2 µg/mL) and CDDP (0.00632 –1.26 µg/mL). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
Conclusions: these nanoconjugates induced apoptosis in MDA-MB-231 cells, probably by both intrinsic and extrinsic pathways, by triggering the oxidative stress mechanisms, and inhibited their
migration potential.
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Procaspase-3/Caspase-3
Procaspase-3Caspase-3
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0 0.316 0.632 0.5 1 0.5 1 0 0.316 0.632 0.5 1 0.5 1
Control CDDP SWCNT-
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SWCNT-
COOH-CDDP
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Control CDDP SWCNT-
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SWCNT-
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Re
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ase-3
/ca
sp
ase-3
(% o
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on
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l)
µg/mL
A. Relative protein expression of (b) procaspase-3/caspase-3 and (c) Bid proteinsafter 24 and 48 h of exposure to 0.5, 1 µg/mL SWCNT-COOH and SWCNT-COOH-
CDDP, 0.316, 0.632 µg/mL CDDP, respectively. In figure (b), the lower graph presents a magnified image of the scale range between 0–250 from the upper
graph. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
µg/mL
32 kDa
43 kDa
24 h
20/17/11 kDa
µg/mL
32 kDa
43 kDa
48 h
20/17/11 kDa
(a)0 0.316 0.632 0.5 0.51 1
0 0.316 0.632 0.5 0.51 1
C CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
C CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
Procaspase-3
β-actin
Cleaved caspase-3
Procaspase-3
β-actin
Cleaved caspase-3
(b)
Cell migration (scratch wound healing assay). (A) Bright-field images presenting the MDA-MB-231 cell culture after 24 and 48 h of wounding and incubation with 0.5, 1 µg/mL SWCNT-COOH and SWCNT-COOH-CDDP, 0.316, 0.632 µg/mL CDDP,
respectively. (B) Quantification of presented images.
o The inhibition of the cell migration was observed after 24 and 48 h of exposure
with 1 µg/mL SWCNT-COOH-CDDP.o A dose of 0.632 µg/mL increased the migration capacity of MDA-MB-231 after
24 h of treatment.
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* **** ***
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Re
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Bid
(%
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Bid
control0.5 µg/mL SWCNT - 0.316 µg/mL CDDP1 µg/mL SWCNT - 0.632 µg/mL CDDP
Control CDDP SWCNT-
COOH
SWCNT-
COOH-CDDPControl CDDP SWCNT-
COOH
SWCNT-
COOH-CDDP
24 h 48 h
22 kDa Bid
22 kDa Bid
(c)
Keywords: breast cancer; carbon nanotubes; cisplatin; apoptosis
B. Relative protein expression of procaspase-8/caspase-8 after 24 and 48 h of exposure to 0.5, 1 µg/mL SWCNT-COOH and SWCNT-COOH-CDDP, 0.316, 0.632 µg/mL CDDP, respectively. ** p < 0.01, *** p < 0.001 vs. control.
o Caspase-3 and caspase-8 were activated in the presence of 1 µg/mL SWCNT-COOH-CDDP starting with 24 h of
treatment. o An increase of procaspase-
3 protein expression was observed after the
treatment with SWCNT-COOH-CDDP.
o The expression of Bid protein was upregulated after 24 h of incubation with nanoconjugates,
followed by a downregulation at 48 h. A
dose with 1 µg/mL SWCNT-COOH-CDDP induced
downregulation of Bid protein expression at both
intervals tested.
References: (1) Collignon, J.; Lousberg, L.; Schroeder, H.; Jerusalem, G. Triple-negative breast cancer: treatment challenges and solutions. Breast Cancer (Dove Med. Press). 2016,8:93-107.(2) Rastogi, V.; Yadav, P.; Bhattacharya, S.S.; Mishra, A.K.; Verma, A.; Pandit, J.K. Carbon nanotubes: An emerging drug carrier for targeting cancer cells. J. Drug Deliv. 2014, 670815.Acknowledgments to PN-III-P2-2.1-PED-2016-0904 project.
A.
B.
