Figure 6. CL4 inhibits tumor growth.

(A) A549 cells (2,000 cells/well in 24-well plates) were treated for 24 h or 48 h with CL4 or CL4sc (200 nM-final concentration) and proliferation was determined by [³H]-thymidine incorporation. Vertical bars indicate the standard deviation values. (B) Growth inhibition of tumors in a mouse xenograft model bearing EGFR-positive A549 cells upon CL4 treatment (57% at 16 days compared to CL4sc control group, P<0.01 by Mann-Whitney test). Day 0 marks the first day of injection. Data are shown as means ± s.e.m. (n = 8 tumors). (C) Three tumors per group selected randomly were excised, lysed, and the pooled lysates were immunoblotted with anti-pEGFR, anti-EGFR, anti-caspase-3, anti-cleaved caspase-8 and anti-αtubulin antibodies, as indicated. Molecular weights of indicated proteins are reported. (D) Cell lysates as in (C) were analyzed by caspase-3 fluorimetric assays. (E) A549 cells (4,000 cells/well in 96-well plates) were left untreated or treated for 24 h with CL4sc (200 nM-final concentration) or CL4 (100 and 200 nM-final concentration) alone or in combination with 1 µM cetuximab or 1 µM gefitinib. Cell viability was analyzed as reported in Methods and was expressed as percent of viable treated cells with respect to control untreated cells (indicated with “C”). Error bars depict means ± s.d. (n = 4). (F) Representative sections of tumors from the CL4sc, CL4sc plus cetuximab, CL4, and CL4 plus cetuximab groups (see “Materials and Methods” for details) stained with H&E, Ki-67 antibody and TUNEL, as indicated. DAPI counterstaining of the boxed regions is shown. Note reduction in cell density in the CL4-treated section stained with H&E. Magnification, 20× for H&E and Ki-67 and 40× for TUNEL and DAPI. (G) Percentage of TUNEL⁺/DAPI⁺ cells, values represent mean ± s.d. for 10 randomly selected fields.