Results

3.1 Gefitinib elicits the cytotoxicity in both low level EGFR and high

We examined the cytotoxicity following treatment with gefitinib in a variety of human cancer cell lines including RKO (colon cancer), A549 (lung cancer), BFTC905 (bladder cancer), MCF7 (breast cancer) and A375 (skin cancer) cells. Gefitinib significantly induced cancer cell death via a concentration-dependent manner in these cancer cell lines (Fig. 1). The order of cytotoxic sensitivity was A375 > MCF7 >

BFTC905 > A549 > RKO cells by exposure to 10-60 μM gefitinib for 24 h. The IC50

value of gefitinib toward cultured human cancer cells lines was 31.45 μM in A375 cells, 40.82 μM in MCF7 cells, 43.49 μM in BFTC905 cells, 83.16 μM in A549 cells, and 91.37 μM in RKO cells.

We have further determined the protein expression of phospho-EGFR and total EGFR in these cencer cell lines. The total protein extracts from various cancer cell lines including RKO, A549, BFTC905, MCF7 and A375 were subjected to western blot analysis. The immunoblot analysis indicated that various human cancer cell lines contained different the protein level of phospho-EGFR and total EGFR (Fig. 2). A375, MCF7 and RKO cells did not significantly express the proteins of the phosphorylated-EGFR and total phosphorylated-EGFR; in contrast, BFTC905 and A549 cells expressed high level of EGFR. Actin was used as a loading control protein. Positive control of phospo-EGFR and EGFR were purified from proteins of A431 cell extracts.

3.2 Gefitinib induces apoptosis in both A375 and BFTC905 cancer cells

Subsequently, we determined the possible involvement of gefitinib in the regulation of cell cycle progression, the effect of gefitinib on A375 and BFTC905 cells was analyzed by flow cytometry (Fig. 3A). Gefitinib did not significantly alter the fractions of G0/G1 and G2/M phases; however, it markedly decreased the fractions of S

phase in both A375 and BFTC905 cells (Fig. 3B). Meanwhile, gefitinib increased the fraction of sub-G1 phase. Treatment with 60 μM gefitinib for 24 h increased apoptosis by 26.6% and 12.3% in A375 and BFTC905 cells, respectively (Fig. 3C).

We further assessed apoptosis from the cells that had been exposed to gefitinib by annexin V and PI staining analysis. The control cells were not significantly stained with fluorochromes; however, the annexin V (+)/PI (−) cells (early apoptosis) and annexin V (+)/PI (+) cells (late apoptosis) were increased by treatment with gefitinib 60 μM for 24 h in both A375 and BFTC905 cells (Fig. 4A). The percentages of apoptosis populations (early and late stages) were quantified. Gefitinib at 60 μM induced about 61.6% of apoptosis in A375 cells and 28.9% of apoptosis in BFTC905 cells (Fig. 4B).

3.3 Gefitinib promotes the hyperpolarization of mitochondria and

The time-lapse observation of cell death was found by gefitinib treatment.

Treatment with gefitinib significantly induced the cell disruption in A375 cells; in contrast, the control group did not induce the cell death (Fig. 5) .

To examine the induction of apoptosis pathway following gefitinib treatment, the cells were subjected to mitochondrial functional assay. Gefitinib induced the hyperpolarization of mitochondrial membrane potential (Fig. 6A). The quantified data showed that treatment with gefitinib for 24 h significantly increased the levels of DiOC6 intensities in a concentration-dependent manner (Fig. 6B). In A375 cells, gefitinib increased the fluorescence intensity of DiOC6 by about 19.1% and 51.0% at 20 μM and 40 μM, respectively.

The apoptosis-regulated proteins were also analyzed by gefitinib. The active form of caspase-3 (17 kDa) protein was induced following treatment with gefitinib for 24 h;

the cleaved form of PARP (89 kDa) protein was also elicited after treatment with gefitinib for 24 h. Actin was used as a loading control protein (Fig. 7).

3.4 Gefitinib inhibits the securin protein expression

To investigate the role of securin on the gefitinib-induced cancer cell death, the securin protein expression after gefitinib-treated A375 cells were analyzed by western blot. The securin protein expression was reduced by gefitinib in A375 cells (Fig. 8A).

Actin was used as a loading control protein. After semi-quantification, gefitinib significantly reduced the protein level of securin in a concentration-dependent manner (Fig. 8B).

Consistently, we examined the effect of gefitinib on the securin protein expression by immunofluorescence staining and confocal microscopy. The β-tubulin and nuclei were stained with the Cy3-labeled anti-β-tubulin and Hoechst 33258, respectively. The intensities of green fluorescence (FITC) exhibited by securin proteins that were markedly decreased after treatment with 40 μM gefitinib for 24 h (Fig. 9).

3.5 Loss of securin enhances the gefitinib-induced cell death and

To determine the role of securin on the cell viability after gefitinib treatment, the securin-wild type and -null HCT116 cancer cells were compared. The cell viability was reduced in both the securin-wild type and -null HCT116 cancer cells following gefitinib treatment with. Treatment with 60 μM gefitinib for 24 h, the cell viability was 18.3%

and 5.1% in securin-wild type and -null cells, respectively. At 60 μM gefitinib, the securin-null cells exhibited greater cytotoxicity than the securin-wild type cells (Fig. 10).

To further determine the role of securin in regulating the gefitinib-induced apoptosis, we transfected a securin-expressed vector (pCT-GFP-sec2). The cell viability was 61.80% and 95.45% in control vector of transfection and securin vector of transfection by treatment with 40 μM gefitinib for 24 h, respectively (Fig. 11).

Overexpression of securin by pCT-GFP-sec2 vector also increased the cell viability in cancer cells. Besides, the transfection of pCT-GFP-sec2 vector was more resistant to the gefitinib-induced cell death than control vector.

3.6 Gefitinib increases the ATF3 protein expression

We had examined the effect of gefitinib on the ATF3 protein expression in cancer cells. Gefitinib increased ATF3 protein expression in A375 cells (Fig. 12A). Actin was used as a loading control protein. The semi-quantified data of western blot showed that gefitinib 40-60 μM for 24 h significantly elevated the protein levels of ATF3 in A375 cells (Fig. 12B).

The expression of ATF3 proteins after treatment with gefitinib were also analyzed by immunofluorescence staining and confocal microscopy. The cytoskeleton and nuclei were stained with BODIPY FL phallacidin and Hoechst 33258, respectively. The red fluorescence (Cy-3) intensity exhibited by ATF3 proteins was increased to translocate to nuclei after treatment with gefitinib 40 μM for 24 h (Fig. 13).

3.7 Gefitinib analogue-1 raises higher cytotoxicity than gefitinib in the

We examined the gefitinib and its analogues on the cytotoxicity in a variety of human cancer cell lines. The cell viability was reduced by treatment with 20 to 80 μM of gefitinib analogue-1 and gefitinib in all cancer cell lines including RKO, A549, BFTC905, MCF7 and A375 cells; however, gefitinib analogue-2 did not significantly induce cytotoxicity in these cancer cells (Fig. 14). Gefitinib analogue-1 was higher on the induction of colon cancer cell death than gefitinib. At the concentrations 60 and 80 μM gefitinib analogue-1 for 24 h in RKO cells, the cell viability was 39.1% and 5.9%, respectively; however, the cell viability for gefitinib at the concentrations 60 and 80 μM was 67.8% and 32.9%, respectively.