Pharmacologic synergy between dual phosphoinositide-3-kinase and mammalian target of rapamycin inhibition and 5-Fluorouracil in PIK3CA mutant gastric cancer cells

Figures & data

Table 1. IC₅₀ values of 5-FU, PI103, and rapamycin, and combination index values of PI103 and 5-FU in gastric cancer cells

		Molecular status			Single agent IC50 (µM)				CI at fu0.5
Cell line		PIK3CA	PTEN		5-FU	PI103	Rapamycin		5-FU+PI103	5-FU+Rapa
AGS		Mutant	Wild-type		79.5 ± 1.8	0.76 ± 0.05	5.82 ± 0.94		0.31 ± 0.03*	0.53 ± 0.02*
HGC27		Mutant	Deleted		11.8 ± 0.2	0.51 ± 0.04	0.49 ± 0.07		0.22 ± 0.02*	0.25 ± 0.01*
IM95		Mutant	Wild-type		37.2 ± 2.7	9.86 ± 0.15	> 100		0.51 ± 0.09*	NA
MKN45		Wild-type	Wild-type		19.3 ± 1.1	1.13 ± 0.09	> 100		1.0 ± 0.01	NA
NUGC4		Wild-type	Wild-type		14.2 ± 2.3	13.61 ± 0.16	> 100		5.65 ± 0.13*	NA

* p < 0.05 compared with value of 1 for additivity. NA, not analyzable.

Figure 1. Effect of PI103, 5-FU and combination on apoptosis induction in gastric cancer cell lines. Nucleosome formation values are displayed as mean ± SD (n = 3) relative to values in untreated control cells. *p < 0.01 compared with controls.

Figure 2. Effect of PI103, 5-FU and the combination on protein expression of E2F1, TS, the PI3K/mTOR pathway, and DNA damage in AGS (A), HGC27 (B), IM95 (C), MKN45 (D) and NUGC4 (E) gastric cancer cells. Cells were exposed to 1x and 3x IC₅₀ concentrations of PI103 and 5-FU, and the combination of PI103 and 5-FU (at IC₅₀ levels, denoted with +) for 24 h, and lysates immunoblotted as described in the Methods section. GAPDH was used as a loading control. A representative image of two independent blots, each using independently prepared cell lysates, is displayed.

Figure 3. (A) Effect of E2F1 siRNA (red) on 5-FU cytotoxicity in gastric cancer cells. (B) Effect of non-growth inhibitory concentration of rapamycin (blue) on 5-FU cytotoxicity in gastric cancer cells. (C) Effect of PIK3CA siRNA (red) on 5-FU cytotoxicity in gastric cancer cells. Charts display mean (± SD) proportion of cells in three independent experiments.

Table 2. Effect of exogenous thymidine on PI103 and 5-FU combination

		Thymidine
Cell line		0 µM	10 µM	100 µM	1 mM
AGS		0.37 ± 0.08*	1.3 ± 0.04	2.4 ± 0.03*	4.5 ± 0.3*
HGC27		0.21 ± 0.01*	1.4 ± 0.02	2.8 ± 0.05*	5.3 ± 0.6*
IM95		0.51 ± 0.08*	1.5 ± 0.02*	3.2 ± 0.07*	6.1 ± 0.4*
MKN45		0.98 ± 0.01	1.9 ± 0.05*	3.9 ± 0.04*	6.4 ± 0.6*
NUGC4		5.1 ± 0.2*	6.8 ± 0.3*	7.2 ± 0.2*	10.2 ± 0.9*

Best fit CI at fu0.5 values are displayed. *p < 0.05 compared with the value of 1 for additivity.

Figure 4. Evaluation of non-growth inhibitory concentration of rapamycin (rapa) and PIK3CA siRNA on 5-FU sensitivity in AGS (A) and HGC27 (B) cells. Charts display mean (± SD) proportion of cells in three independent experiments. NTC, non-treated control.

Figure 5. (A) Tumor sizes of mouse xenograft models of HGC27 cells treated with PI103, 5-FU, the combination and control (10% DMSO). The chart displays mean (± SD) size of tumors (n = 3). (B) Western immunoblot showing effect of PI103, 5-FU and the combination on levels of E2F1, TS, the PI3K/mTOR pathway proteins and H2AX in HGC27 xenograft tumor samples. A representative of two independent blots from independently prepared tissue homogenates is displayed.