World J Oncol

World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access

Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc

Journal website http://www.wjon.org

Original Article

Volume 4, Number 1, February 2013, pages 26-36

Usefulness of Daily Fractionated Administration of Wortmannin Combined With γ-Ray Irradiation in Terms of Local Tumor Response and Lung Metastasis

Figure 1. Cell survival curves for the total cell population from B16-BL6 tumors irradiated with γ-rays following single or four intraperitoneal administrations of wortmannin in combination with nicotinamide treatment or mild temperature hyperthermia (MTH) on day 18 after tumor cell inoculation. Circles, γ-ray irradiation only; Squares, γ-ray irradiation with nicotinamide treatment; Triangles, γ-ray irradiation with MTH. Bars represent standard errors (n = 9).

Figure 2. Dose response curves of the net micronucleus frequency for total (open symbols, left panels) and quiescent (solid symbols, right panels) cell populations from B16-BL6 tumors irradiated with γ-rays following single or four intraperitoneal administrations of wortmannin in combination with nicotinamide treatment or mild temperature hyperthermia (MTH) on day 18 after tumor cell inoculation. Circles, γ-ray irradiation only; Squares, γ-ray irradiation with nicotinamide treatment; Triangles, γ-ray irradiation with MTH. Bars represent standard errors (n = 9).

Figure 3. Cell survival curves for the total cell population from B16-BL6 tumors irradiated with γ-rays following single or four intraperitoneal administrations of wortmannin in combination with nicotinamide treatment or mild temperature hyperthermia (MTH) on day 18 after tumor cell inoculation. Circles, γ-ray irradiation only; Squares, γ-ray irradiation with nicotinamide treatment; Triangles, γ-ray irradiation with MTH. Bars represent standard errors (n = 9).

**Table 1.** Surviving Fraction and Micronucleus Frequency at 0 Gy
	Without wortmannin	With wortmannin (once)	With wortmannin (4 times)
^a: Mean ± standard error (n = 9). Q cells showed significantly higher micronucleus frequencies than the total cell population under each set of conditions (P < 0.05). Wortmannin administration resulted in significantly lower surviving fractions and significantly higher micronucleus frequencies in both the total and Q cell populations than absolutely no treatment (P < 0.05).
Surviving fraction at 0 Gy (%)
Without nicotinamide or mild temperature hyperthermia	84.4 ± 8.2^a	69.8 ± 6.3	60.0 ± 5.5
With nicotinamide	81.4 ± 7.3	60.0 ± 5.7	55.0 ± 4.3
With mild temperature hyperthermia	83.5 ± 8.7	60.5 ± 5.3	56.0 ± 5.9
Micronucleus frequency at 0 Gy
Total tumor cell population
Without nicotinamide or mild temperature hyperthermia	0.050 ± 0.005	0.070 ± 0.007	0.081 ± 0.008
With nicotinamide	0.057 ± 0.006	0.082 ± 0.008	0.089 ± 0.008
With mild temperature hyperthermia	0.054 ± 0.005	0.080 ± 0.008	0.085 ± 0.009
Quiescent cells
Without nicotinamide or mild temperature hyperthermia	0.077 ± 0.007	0.094 ± 0.009	0.102 ± 0.01
With nicotinamide	0.084 ± 0.008	0.101 ± 0.01	0.11 ± 0.01
With mild temperature hyperthermia	0.081 ± 0.008	0.099 ± 0.009	0.103 ± 0.01

Table 1. Surviving Fraction and Micronucleus Frequency at 0 Gy

Without wortmannin

With wortmannin (once)

With wortmannin (4 times)

^a: Mean ± standard error (n = 9). Q cells showed significantly higher micronucleus frequencies than the total cell population under each set of conditions (P < 0.05). Wortmannin administration resulted in significantly lower surviving fractions and significantly higher micronucleus frequencies in both the total and Q cell populations than absolutely no treatment (P < 0.05).

Surviving fraction at 0 Gy (%)

Without nicotinamide or mild temperature hyperthermia

84.4 ± 8.2^a

69.8 ± 6.3

60.0 ± 5.5

With nicotinamide

81.4 ± 7.3

60.0 ± 5.7

55.0 ± 4.3

With mild temperature hyperthermia

83.5 ± 8.7

60.5 ± 5.3

56.0 ± 5.9

Micronucleus frequency at 0 Gy

Total tumor cell population

Without nicotinamide or mild temperature hyperthermia

0.050 ± 0.005

0.070 ± 0.007

0.081 ± 0.008

With nicotinamide

0.057 ± 0.006

0.082 ± 0.008

0.089 ± 0.008

With mild temperature hyperthermia

0.054 ± 0.005

0.080 ± 0.008

0.085 ± 0.009

Quiescent cells

Without nicotinamide or mild temperature hyperthermia

0.077 ± 0.007

0.094 ± 0.009

0.102 ± 0.01

With nicotinamide

0.084 ± 0.008

0.101 ± 0.01

0.11 ± 0.01

With mild temperature hyperthermia

0.081 ± 0.008

0.099 ± 0.009

0.103 ± 0.01

**Table 2.** Enhancement Ratios^a due to Combined Treatment With Wortmannin
	With wortmannin (once)	With wortmannin (4 times)
^a: The ratio of the dose of radiation necessary to obtain each end-point without wortmannin to that needed to obtain each end-point with wortmannin; ^b: Mean ± standard error (n = 9); Letters^c-e represent significant differences between two values (P < 0.05).
Surviving fraction = 0.03
Total cell population	1.05 ± 0.05^{b, c}	1.2 ± 0.1^c
Net micronucleus frequency = 0.6
Total cell population	1.05 ± 0.05^{d, e}	1.2 ± 0.1^d
Quiescent cells	1.2 ± 0.1^e	1.3 ± 0.1

Table 2. Enhancement Ratios^a due to Combined Treatment With Wortmannin

With wortmannin (once)

With wortmannin (4 times)

^a: The ratio of the dose of radiation necessary to obtain each end-point without wortmannin to that needed to obtain each end-point with wortmannin; ^b: Mean ± standard error (n = 9); Letters^c-e represent significant differences between two values (P < 0.05).

Surviving fraction = 0.03

Total cell population

1.05 ± 0.05^{b, c}

1.2 ± 0.1^c

Net micronucleus frequency = 0.6

Total cell population

1.05 ± 0.05^{d, e}

1.2 ± 0.1^d

Quiescent cells

1.2 ± 0.1^e

1.3 ± 0.1

**Table 3.** Enhancement Ratios^a due to Combined Treatment With Nicotinamide, or Mild Temperature Hyperthermia
	Nicotinamide	Mild temperature hyperthermia
^a: The ratio of the dose of radiation necessary to obtain each end-point without nicotinamide or mild temperature hyperthermia to that needed to obtain each end-point with nicotinamide or mild temperature hyperthermia; ^b: Mean ± standard error (n = 9).
Surviving fraction = 0.03
Total cell population
Without wortmannin	1.25 ± 0.1^b	1.1 ± 0.05
With wortmannin (once)	1.2 ± 0.1	1.1 ± 0.05
With wortmannin (4 times)	1.05 ± 0.05	1.1 ± 0.05
Net micronucleus frequency = 0.6
Total cell population
Without wortmannin	1.2 ± 0.1	1.1 ± 0.05
With wortmannin (once)	1.2 ± 0.1	1.05 ± 0.05
With wortmannin (4 times)	1.05 ± 0.05	1.1 ± 0.05
Quiescent cells
Without wortmannin	1.1 ± 0.05	1.2 ± 0.1
With wortmannin (once)	1.05 ± 0.05	1.15 ± 0.05
With wortmannin (4 times)	1.05 ± 0.05	1.15 ± 0.05

Table 3. Enhancement Ratios^a due to Combined Treatment With Nicotinamide, or Mild Temperature Hyperthermia

Nicotinamide

Mild temperature hyperthermia

^a: The ratio of the dose of radiation necessary to obtain each end-point without nicotinamide or mild temperature hyperthermia to that needed to obtain each end-point with nicotinamide or mild temperature hyperthermia; ^b: Mean ± standard error (n = 9).

Surviving fraction = 0.03

Total cell population

Without wortmannin

1.25 ± 0.1^b

1.1 ± 0.05

With wortmannin (once)

1.2 ± 0.1

1.1 ± 0.05

With wortmannin (4 times)

1.05 ± 0.05

1.1 ± 0.05

Net micronucleus frequency = 0.6

Total cell population

Without wortmannin

1.2 ± 0.1

1.1 ± 0.05

With wortmannin (once)

1.2 ± 0.1

1.05 ± 0.05

With wortmannin (4 times)

1.05 ± 0.05

1.1 ± 0.05

Quiescent cells

Without wortmannin

1.1 ± 0.05

1.2 ± 0.1

With wortmannin (once)

1.05 ± 0.05

1.15 ± 0.05

With wortmannin (4 times)

1.05 ± 0.05

1.15 ± 0.05

**Table 4.** Dose-Modifying Factors for Quiescent Cells Relative to the Total Tumor Cell Population^a
	Without wortmannin	With wortmannin (once)	With wortmannin (4 times)
^a: The ratio of the dose of radiation necessary to obtain each end-point in the quiescent cell population to that needed to obtain each end-point in the total tumor cell population; ^b: Mean ± standard error (n = 9).
Net micronucleus frequency = 0.6
γ-Rays only	1.7 ± 0.15^b	1.5 ± 0.1	1.6 ± 0.15
With nicotinamide	1.8 ± 0.15	1.65 ± 0.15	1.7 ± 0.15
With mild temperature hyperthermia	1.6 ± 0.15	1.4 ± 0.1	1.55 ± 0.1

Table 4. Dose-Modifying Factors for Quiescent Cells Relative to the Total Tumor Cell Population^a

Without wortmannin

With wortmannin (once)

With wortmannin (4 times)

^a: The ratio of the dose of radiation necessary to obtain each end-point in the quiescent cell population to that needed to obtain each end-point in the total tumor cell population; ^b: Mean ± standard error (n = 9).

Net micronucleus frequency = 0.6

γ-Rays only

1.7 ± 0.15^b

1.5 ± 0.1

1.6 ± 0.15

With nicotinamide

1.8 ± 0.15

1.65 ± 0.15

1.7 ± 0.15

With mild temperature hyperthermia

1.6 ± 0.15

1.4 ± 0.1

1.55 ± 0.1

**Table 5.** The Numbers of Metastases From the Irradiated Tumors That Received Cytotoxic Treatment Producing a Similar Initial Local Effect^a
	Without wortmannin	With wortmannin (once)	With wortmannin (4 times)
^a: Based on the data shown in Figure 3, the estimated numbers of lung metastatic nodules from local tumors that received γ-ray irradiation with or without wortmannin in combination with nicotinamide or mild temperature hyperthermia, which produced an identical surviving fraction of 0.03 as an initial effect on Figure 1;^b: Mean ± standard error (n = 9); Letters^c-h represent significant differences between two values (P < 0.05).
Surviving fraction = 0.03
γ-Rays only	14.2 ± 1.4^{b, c, d, e}	12.4 ± 1.3	12.0 ± 1.2
With nicotinamide	13.3 ± 1.3	11.5 ± 1.2^{c, f}	11.4 ± 1.2^{d, g}
With mild temperature hyperthermia	14.2 ± 1.4^{f, g, h}	12.2 ± 1.2	10.7 ± 1.1^{e, h}

Table 5. The Numbers of Metastases From the Irradiated Tumors That Received Cytotoxic Treatment Producing a Similar Initial Local Effect^a

Without wortmannin

With wortmannin (once)

With wortmannin (4 times)

^a: Based on the data shown in Figure 3, the estimated numbers of lung metastatic nodules from local tumors that received γ-ray irradiation with or without wortmannin in combination with nicotinamide or mild temperature hyperthermia, which produced an identical surviving fraction of 0.03 as an initial effect on Figure 1;^b: Mean ± standard error (n = 9); Letters^c-h represent significant differences between two values (P < 0.05).

Surviving fraction = 0.03

γ-Rays only

14.2 ± 1.4^{b, c, d, e}

12.4 ± 1.3

12.0 ± 1.2

With nicotinamide

13.3 ± 1.3

11.5 ± 1.2^{c, f}

11.4 ± 1.2^{d, g}

With mild temperature hyperthermia

14.2 ± 1.4^{f, g, h}

12.2 ± 1.2

10.7 ± 1.1^{e, h}