Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study

<div><p>Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively targets cancer cells. The present preclinical study investigated the anti-cancer efficiency of ONC201, a first-in-class small molecule TRAIL inducer, in lung cancer cells. We showed that ONC201 was cytotoxic and anti-proliferative in both established (A549 and H460 lines) and primary human lung cancer cells. It was yet non-cytotoxic to normal lung epithelial cells. Further, ONC201 induced exogenous apoptosis activation in lung cancer cells, which was evidenced by TRAIL/death receptor-5 (DR5) induction and caspase-8 activation. The caspase-8 inhibitor or TRAIL/DR5 siRNA knockdown alleviated ONC201’s cytotoxicity against lung cancer cells. Molecularly, ONC201 in-activated Akt-S6K1 and Erk signalings in lung cancer cells, causing Foxo3a nuclear translocation. For the <i>in vivo</i> studies, intraperitoneal injection of ONC201 at well-tolerated doses significantly inhibited xenografted A549 tumor growth in severe combined immunodeficient (SCID) mice. Further, ONC201 administration induced TRAIL/DR5 expression, yet inactivated Akt-S6K1 and Erk in tumor tissues. These results of the study demonstrates the potent anti-lung cancer activity by ONC201.</p></div

ONC201 provokes apoptosis in human lung cancer cells.

<p>A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative caspase-8/-3 activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. *<b><i>p</i></b><0.05 vs “C” group.</p

Inhibition of extrinsic apoptosis activation attenuates ONC201’s cytotoxicity in human lung cancer cells.

<p>A549 cells transfected with scramble control siRNA (SCR-siRNA), TRAIL siRNA or DR5 siRNA (100 nM each) were treated with ONC201 (10 μM) for applied time, TRAIL/DR5 mRNA and protein expressions were shown (A, GAPDH was tested as the control); Cell viability and apoptosis were examined by MTT assay (B) and ssDNA apoptosis ELISA assay (C), respectively. A549 cells were pre-treated with z-IETD-fmk (40 μM) or z-VAD-fmk (40 μM) for 1 hour, followed by ONC201 (10 μM) treatment for indicated time, cell viability and apoptosis were examined by MTT assay (D) and Sub G1 FACS assay (E), respectively. H460 cells or primary human lung cancer cells (“Pat-2”), pretreated with z-IETD-fmk (40 μM, 1 hour) or TRAIL plus DR5 siRNA (100 nM each, 36 hours), were treated with ONC201 (10 μM) for 72 hours, cell viability was tested by MTT assay (F). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. “Veh” stands for 0.2% DMSO (D and E). *<b><i>p</i></b><0.05 vs “No siRNA” group (A). *<b><i>p</i></b><0.05 vs “SCR siRNA” group (B and C). *<b><i>p</i></b><0.05 vs “Veh” group (D and E). *<b><i>p</i></b><0.05 vs ONC201 only group (F).</p

Intraperitoneal injection of ONC201 inhibits A549 xenograft tumor growth in SCID mice.

<p>SCID mice bearing A549 tumors (n = 10) were administrated with vehicle control (“Saline”), or ONC201 (10/50 mg/kg, <i>i</i>.<i>p</i>.), tumor volumes (in mm³, A) and mice body weights (in grams, C) were recorded every week for a total of 5 weeks. The estimated daily tumor growth (in mm³ per day, B) was also presented. The signaling molecule in the xenografted tumor tissues (3 days post initial ONC201 treatment) were tested by Western blot assay (D) and IHC staining (E, for p-Akt). * <b><i>p</i></b> < 0.05 vs. group of Vehicle control. ** <b><i>p</i></b> < 0.05 vs. group of ONC201 at 10 mg/kg. The above xenograft experiments were repeated twice, and similar results were obtained. Bar = 100 μm (E).</p

ONC201 induces death in human lung cancer cells.

<p>A549 (A-C), H460 (D) or primary human lung cancer cells (“Pat-1/-2”) (D), as well as the lung epithelial BEAS-2B cells (D) and human HL-7702 hepatocytes (E) were treated with applied concentration of ONC201 for indicated time, cells were subjected to MTT assay (A, D and E), colony formation assay (B) and LDH release assay (C). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. *<b><i>p</i></b><0.05 vs “C” group.</p