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Combination of DSF or DSF/Cu 2+ attenuate IR-induced G2/M phase arrest

5. Results

5.18 Combination of DSF or DSF/Cu 2+ attenuate IR-induced G2/M phase arrest

39

Fig. 9: Combination with DSF or DSF/Cu2+ and cisplatin in HNSCC cell lines.

(A) Cells were treated with various concentrations of cisplatin and 5 µM DSF for 72 h and the cytotoxic effect was determined by MTT assay. (B) Cells were treated with DSF (5 µM), DSF/Cu2+ (0.1 µM), cisplatin (UM-SCC9 and UM-SCC47: 0.3 µM; UT-SCC33: 0.6 µM) or a combination of both for 48 h. The cell cycle distribution was detected by flow cytometry. The numbers in the graph represent proportions as a percentage of sub-G1 (<2N); G1 (2N); S-phase (S); G2/M phase (4N); and aneuploid cells (>4N). (C) The percentage of cells in the G2/M phase is compared. ##P<0.01: cisplatin vs. control; *P<0.05: cisplatin vs. cisplatin+DSF or cisplatin+DSF/Cu2+; one-way ANOVA.

40

To explore the potential nature of DSF or DSF/Cu2+ sensitized cells to IR, cell cycle distribution analysis was performed after combination exposure. Cells were pre-treated with 5 μM DSF or 0.1 μM DSF/Cu2+ following exposure at 10 Gy IR. As shown in Figure 10D and 10E, a dramatic G2/M phase activation was detected after 48 h of IR with an increase from 27.7% to 53.6% in UM-SCC9 and from 28.2% to 50.0% in UM-SCC47, respectively, with a concomitant decrease in the G1 phase as well. However, when combined with DSF or DSF/Cu2+, it resulted in reducing the G2/M phase from 53.6% to 40.2% and 41.9% in UM-SCC9, and from 50.0% to 39.5% and 39.7% in UM-SCC47, respectively. Taken together, these results provide strong evidence that DSF or DSF/Cu2+ could attenuate IR-induced G2/M phase arrest where checkpoints in the cell cycle or DNA repair signaling pathways might be involved.

41

UT-SCC11BUM-SCC9UM-SCC47

Control 2 Gy 4 Gy 8 Gy 20 Gy

G2/M 27.7

G2/M 30.6

G2/M 24.0

G2/M 27.8

G2/M 29.2

G2/M 35.8

G2/M 45.4

G2/M 62.4 G2/M

40.8 G2/M

28.2

G2/M 29.6

G2/M 27.3

G2/M 35.0

G2/M 63.1 G2/M

41.9 10 Gy

G2/M 36.6

G2/M 48.8 G2/M

42.3

FL2 (DNA Content)

Cell Counts

C

A B

0 0.03 0.1

0.3 1 3 10 30 0

50 100

DSF Concentration (M)

Viability (%)

0

0.03 0.1

0.3 1 0

50

100 0 Gy

10 Gy

DSF/Cu2+ Concentration (M)

0 2 4 6

0.001 0.01 0.1

1 IR

IR+DSF IR+DSF/Cu2+

Dose (Gy)

Surviving Fraction

42

Fig. 10: Radiosensitizing effect of DSF or DSF/Cu2+ in HNSCC cell lines.

(A) Cells were pre-treated with various concentrations of DSF or DSF/Cu2+ and then irradiated with 10 Gy. After 72 h, viability was analyzed using MTT assay. (B) Cells were pre-treated with DSF (1 µM) or DSF/Cu2+ (0.1 µM) and then irradiated with 2-6 Gy. After 24 h, cells were reseeded in drug-free medium for 9-12 days. The surviving fraction at different dosages of IR was compared using the LQ-Model formula. (C) Cells were treated with the indicated dosages of IR. Cell cycle distributions were detected using flow cytometry 48 h later. The numbers in the graph represent proportions as a percentage of sub-G1 (<2N); G1 (2N); S-phase (S); G2/M phase (4N); and aneuploid cells (>4N). (D) Cells were pre-treated with DSF (5 µM) or DSF/Cu2+ (0.1 µM) and then irradiated with 10 Gy. 48 h later, the levels of each cell cycle phase were measured using flow cytometry. The numbers in the graph represent proportions as a percentage of sub-G1 (<2N); G1 (2N); S-phase (S); G2/M phase (4N); and aneuploid cells (>4N). (E) The percentage of cells in G2/M phase is compared. ##P<0.01: IR vs. control; *P<0.05: IR vs. IR+DSF or IR+DSF/Cu2+; one-way ANOVA.

E D

G2/M 27.7

G2/M 28.2

G2/M 29.0

G2/M 27.8

G2/M 28.4

G2/M 53.6

G2/M 28.6

G2/M 39.5

G2/M 41.9 G2/M

40.2

UM-SCC9UM-SCC47

Control DS F DS F/Cu2+ IR IR+DS F IR+DS F/Cu2+

G2/M 39.7 G2/M

50.0

FL2 (DNA Content)

Cell Counts

UM-SCC9

control DSF 2+

DSF/Cu IR IR+DSF

2+

IR+DSF /Cu 0

20 40

60 ##

* *

Cells in G2/M Phase (%) UM-SCC47

control DSF 2+

DSF/Cu IR IR+DSF

2+

IR+DSF /Cu 0

20 40 60

##

* *

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5.19 Cytotoxic effect by the triple treatment of DSF or DSF/Cu2+, cisplatin and IR in HNSCC cell lines

In previous experiments, we proved that DSF or DSF/Cu2+ enhanced the cytotoxic effect of cisplatin and IR by abolishing the G2/M phase arrest in cell cycle. We next investigated the effect of triple exposure on the combination of DSF or DSF/Cu2+, cisplatin, and IR. As shown in Figure 11A and 11B, combining 5 μM DSF or 0.1 μM DSF/Cu2+ with 2.5 μM cisplatin increased apoptosis to 34.71% and 26.69% in UM-SCC9, 31% and 25.48% in UM-SCC47, 37.55%, and 31.77% in UM-SCC11B, respectively, which indicated the induction of remarkably higher cell deaths compared to each treatment alone. Furthermore, the combination with IR indicated a significant enhancement of apoptotic cell death. A cytotoxic effect was detected in IR+cisplatin+DSF and IR+cisplatin+DSF/Cu2+ with 42.04% and 32.21%in UM-SCC9, 43.9%

and 31.91% in UM-SCC47, 45.37% and 38.08% in UM-SCC11B, respectively. Collectively, this effect of suppressing cell growth and inducing apoptosis in triple combination provides a promising clinical treatment strategy to achieve a better chemo-radio-sensitizing effect in HNSCC.

Fig. 11: DSF or DSF/Cu2+ combined with cisplatin and IR enhance apoptosis in HNSCC cell lines.

(A) Cells were pre-treated with DSF (5 µM), DSF/Cu2+ (0.1 µM), cisplatin (2.5 µM), or a combination of both, then exposed to IR (10 Gy). After 48 h, apoptosis was detected using flow cytometry. (B) Graphical representation of the statistical analysis. **P<0.01: cisplatin+DSF vs.

cisplatin or DSF; *P<0.05: cisplatin+DSF/Cu2+ vs. cisplatin or DSF/Cu2+; #P<0.05 or ##P<0.01:

IR+cisplatin+DSF vs. cisplatin+DSF or IR+cisplatin+DSF/Cu2+ vs. cisplatin+DSF/Cu2+, one-way ANOVA.

B

UM-SCC9

control DSF

2+

DSF /Cu

Cisplatin IR Cisplatin+DSF

2+

Cisplatin+DSF /Cu

IR+

Cisplatin+DSF 2+

IR+

Cis platin+DSF

/Cu 0

20 40 60

**

*

##

##

Apoptosis (%)

UM-SCC47

control DSF

2+

DSF /Cu

Cis platin IR

Cis platin+DSF

2+

Cisplatin+DSF /Cu

IR+

Cis platin+DSF

2+

IR+

Cisplatin+DSF /Cu 0

20 40 60

**

*

#

#

UM-SCC11B

control DSF

2+

DSF /Cu

Cis platin IR

Cis platin+DSF

2+

Cisplatin+DSF /Cu

IR+

Cis platin+DSF

2+

IR+

Cisplatin+DSF /Cu 0

20 40 60

** *

##

#

44

UM-SCC9UM-SCC47UM-SCC11B

FL1 (Annexin-V)

FL3 (PI)

Control DSF DSF/Cu2+ Cisplatin IR

Cisplatin+DSF Cisplatin+DSF/Cu2+ IR Cisplatin+DSF IR Cisplatin+DSF/Cu2+

Control DSF DSF/Cu2+ Cisplatin IR

Cisplatin+DSF Cisplatin+DSF/Cu2+ IR Cisplatin+DSF IR Cisplatin+DSF/Cu2+

Control DSF DSF/Cu2+ Cisplatin IR

Cisplatin+DSF Cisplatin+DSF/Cu2+ IR Cisplatin+DSF IR Cisplatin+DSF/Cu2+

A

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5.20 Treatment with DSF or DSF/Cu2+, cisplatin, and IR induces ROS generation in HNSCC cell lines

To gain a better understanding of the cytotoxicity in this triple treatment, we explored the generation of ROS after exposure to DSF or DSF/Cu2+, cisplatin and IR. As shown in Figure 12A and 12B, the combination 5 μM DSF with 2.5 μM cisplatin enhanced ROS activity to 38.7% in UM-SCC9, 35.8% in UM-SCC47, 35.9% in UM-SCC11B, respectively, compared to each treatment alone. IR also plays a core role in the accumulation of ROS. When combined with 10 Gy IR, addition of cisplatin and DSF or DSF/Cu2+, further increasing ROS activity was observed with 46.3% and 37.4% in UM-SCC9, 44.0% and 32.5% in UM-SCC47, 46.3% and 34.7% in UM-SCC11B, respectively, compared to unirradiated samples. In conclusion, this evidence proves that the triple treatment of DSF or DSF/Cu2+, cisplatin and IR substantially enhance the ROS generation, which is responsible for the cytotoxic effect and might be emphasized as a potential method in HNSCC treatment.

Fig. 12: DSF or DSF/Cu2+ combined with cisplatin and IR induce ROS generation in HNSCC cell lines.

(A) Cells were pre-treated with DSF (5 µM), DSF/Cu2+ (0.1 µM), cisplatin (2.5 µM), or a combination of both, then exposed to IR (10 Gy). ROS activity was detected 24 h later using flow cytometry. The numbers in the graph represent the ROS activity. (B) Graphical representation of the statistical analysis of ROS activity. **P<0.01: cisplatin+DSF vs. cisplatin or DSF, #P<0.05 or ##P<0.01: IR+cisplatin+DSF vs. cisplatin+DSF, or IR+cisplatin+DSF/Cu2+ vs.

cisplatin+DSF/Cu2+, one-way ANOVA.

B

UM-SCC9

control DSF 2+

DSF/Cu Cispl

atin IR Cispl

atin+D SF 2+

Cispl atin+DSF/

Cu

IR+C ispl

atin+

DSF 2+

IR+C ispl

atin+

DSF/

Cu 0

20 40 60

**

##

##

ROS Activity (%)

UM-SCC47

control DSF 2+

DSF/Cu Cispl

atin IR Cispl

atin+DSF 2+

Cispl atin+DSF/

Cu

IR+Cispl atin+

DSF 2+

IR+Cispl atin+

DSF/

Cu 0

20 40 60

**

#

#

UM-SCC11B

control DSF 2+

DSF/Cu Cispl

ain IR Cispl

atin+DSF 2+

Cispl atin+DSF/

Cu

IR+Cispl atin+

DSF 2+

IR+Cispl atin+

DSF/

Cu 0

20 40 60

**

##

#

46

UM-SCC9UM-SCC47UM-SCC11B

FL2 (ROS Activity)

Cell Counts

Control DSF DSF/Cu2+ Cisplatin IR

Cisplatin+DSF IR Cisplatin+DSF

Control DSF DSF/Cu2+ Cisplatin IR

Cisplatin+DSF IR Cisplatin+DSF

Control DSF DSF/Cu2+ Cisplatin IR

Cispaltin+DSF

Cisplatin+DSF/Cu2+

Cisplatin+DSF/Cu2+

IR Cisplatin+DSF/Cu2+

IR Cisplatin+DSF/Cu2+

Cisplatin+DSF/Cu2+ IR Cisplatin+DSF/Cu2+

IR Cisplatin+DSF

26.5 28.4 26.8

38.7

26.1

25.0

31.2

32.0

46.3 37.4

20.9 23.5 20.0 27.1

35.8 28.4 44.0

27.6

32.5

26.3 17.4

35.9 30.4

19.6 23.2

34.7 46.3

A