NVP-BEZ-235 enhances radiosensitization via blockade of the PI3K/mTOR pathway in cisplatin-resistant non-small cell lung carcinoma

Abstract

Introduction: Most drug resistant cancer cells also develop resistance to radiation therapy. In this study, we hypothesized that the dual inhibitor of phosphatidylinositol-3 kinase/mammalian target of rapamycin, NVP-BEZ-235, could potentially enhance radiosensitization in cisplatin-resistance (CDDP-R) non-small cell lung cancer (NSCLC) cells by disabling autophagy as a mechanism of self-preservation.

Methods: We used both in vitro and in vivo approaches, including clonogenic assays, Western blotting, molecular analyses of autophagy and apoptosis, a xenograft model of tumor growth, and immunohistochemical analysis.

Results: Basal p-Akt, p-mTOR and p-S6R proteins were enhanced in CDDP-R NSCLC cells. CDDP-R-resistant NSCLC cells are less radiation sensitive in comparison to parental cells (DER=0.82, p=0.02); BEZ-235 enhanced the radiosensitivity (DER=1.2, p=0.01). In addition, combining BEZ-235/RT showed a dramatic tumor growth delay in a mouse xenograft model. Immunohistochemistry showed that combination therapy yielded 50% decrease in caspase-3 activity. Moreover, cell proliferation was reduced by 87.8% and vascular density by 86.1%. These results were associated with a downregulation of PI3K/mTOR signaling pathway and an increase in autophagy.

Conclusions: These findings may be utilized as a novel strategy to enhance the efficacy of radiation therapy in drug-selected non-small cell lung cancer exhibiting radioresistance.

Keywords: NSCLC; autophagy; cisplatin resistance; mTOR; radiosensitization.

Figure 1. Cisplatin-resistant H460-luc2 cells show an increased expression of stem cell markers and increased activity of the AKT-mTOR pathway

Cisplatin resistant (CDDP-R) H460-luc2 cells show an increase in the phosphorylation of AKT, mTOR, and the S6 ribosomal subunit, without a change in total protein expression.

Figure 2. BEZ-235 treatment sensitizes CDDP-R H460-luc2 cells to cisplatin via inhibition of the PI3K/Akt/mTOR pathway

Treatment of cisplatin-resistant H460-luc2 cells with concurrent BEZ-235 and cisplatin decreases cell survival. Cells were treated with DMSO (control), 3μM cisplatin, 50nm BEZ-235, or both (A). The parental cell line was susceptible to cisplatin treatment; neither cell line was significantly affected by BEZ-235 alone. Combining cisplatin and BEZ-235 reduced survival in both the parental and cisplatin-resistant cell lines. Treatment with 50nM BEZ-235 eliminates ribosomal S6 phosphorylation in both cisplatin-resistant H460-luc2 cells and the parental cell line (B). Both total and phosphorylated AKT were decreased in both cell lines, but the effect was more pronounced in cisplatin-resistant cells (C).

Figure 3. BEZ-235 significantly enhances the radiosensitivity of CDDP-R H460-Luc2 and parental cells via the induction of autophagy

Parental or cisplatin-resistant cells were pretreated with 50nM BEZ-235 or DMSO for 24 hours, followed by irradiation with 0, 2, 4 or 6Gy (A). Untreated CDDP-resistant cells show greater resistance to radiation than the parental cell line (p=0.006). Treatment with BEZ-235 greatly increases radiation sensitivity in both the parental and CDDP-resistant cell lines (p=0.005 and p=0.007); this effect is greater in the radiation-resistant cell line (DER=1.5 vs 1.8). The difference in survival is much higher for the CDDP-resistant cells (p=0.007). Treatment with 50nm BEZ-235 for 2 hours and subsequent administration of 5Gy radiation induces autophagy in CDDP-R H460-Luc2 cells (B). Both parental and radiation-resistant cell lines treated with either BEZ-235 or radiation had increased levels of autophagy; treatment with BEZ-235 was more effective in both cell lines. Combined radiation and BEZ-235 treatment resulted in a significant enhancement in the levels of autophagy in both cell lines. In the parental cell line, this effect was additive; in CDDP-R cells these treatments acted synergistically, resulting in a significant increase in measurable levels of autophagy. Parental H460-Luc2 cells exhibited increased cleaved caspase-3 expression following treatment with RT, BEZ-235 + RT, 3μM cisplatin, or cisplatin + RT (C). The highest increased in expression was seen in cells treated with BEZ-235 and cisplatin; the cisplatin alone group showed the next highest level. BEZ-235 + RT induced less cleaved caspase-3 than RT alone. In CDDP-R cells, RT alone resulted caspase-3 cleavage (D). Co-treatment with BEZ-235 and RT, BEZ-235 and cisplatin, or treatment with cisplatin alone did not result in any measurable apoptosis, as determined by cleaved-caspase 3 expression.

Figure 4. Treatment with BEZ-235 and RT delays tumor growth

Tumors consisting of cisplatin-resistant cells grew more rapidly than tumors derived from the parental cell line when animals were untreated. BEZ-235 treatment delays tumor growth more than RT. Combination therapy, BEZ-235 + RT, significantly delays tumor growth.

Figure 5. RT, BEZ-235, and combination therapy alter expression of caspase-3, KI67, p62, and von Willebrand factor in cisplatin-resistant cells

Untreated CDDP-R cells produce slightly less caspase-3 than the parental cells (A, B; U). Caspase-3 is increased slightly after BEZ-235 treatment (C, U) and significantly after irradiation (D, U); combinatory treatment shows an intermediate effect (E, U). Cell proliferation (as measured by KI67 staining) was decreased by both BEZ-235 (58.2%; H, V) and RT (70.3%; I, V), and combination treatment (87.8%; J, V). Confirmation of this decrease was obtained after staining for p62. p62 expression is decreased by BEZ-235 (50%; M, W), RT (35.4%; N, W), and combination therapy (74.5%; O, W). Finally, angiogenic potential was assessed by staining for von Willebrand's factor. CDDP-R cells produce slightly more vWF than the parental cells at baseline (Figure 6P, Q). BEZ-235 decreases vWF expression by 75% (R, X), whereas RT decreases vWF expression in CDDP-R cells 48% (S, X). Combining radiation and BEZ-235 decreases vWF expression 86.1% (T, X).