TNFa inhibitor C87 sensitizes EGFRvIII transfected glioblastoma cells to gefitinib by a concurrent blockade of TNFa signaling

Gefitinib inhibition triggers an adaptive response in glioblastoma cells. (A) The band intensity of immunoblots in Figure 1C were quantified (mean ± s.d., n = 3 independent experiments, Student’s t-test). (B) U87vIII cells were treated with 2 µM gefitinib for the indicated time intervals, and total Akt, phospho-Akt, total STAT3, phospho-STAT3, total p38 MAPK and phospho-p38 MAPK were detected by immunoblotting.

High levels of TNFa in the glioblastoma microenvironment contributed to the primary resistance to EGFR inhibition. After TNFa (1 ng/mL) alone, or gefitinib (25 µM) alone, or combination of the two treated U87vIII (A) or LN229vIII (B) cells for 48 hours, cell viability was tested by MTT assay. (C) The relative expression of TNFa mRNA in 15 low grade astrocytoma (WHO II-III) tissues and 16 glioblastoma tissues were detected with qRT-PCR method. In (A), (B), and (C), data were mean ± SEM, n = 3 independent experiments, Student’s t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (D) Top: Immunoblotting analysis of protein levels of TNFa in the 15 low grade astrocytoma tissues (WHO II-III, serial number 1#-15#) and 16 glioblastoma tissues (serial number 16#-31#). Bottom: Western blot band intensity of TNFa was quantified and normalized to the internal control (mean ± SD, n = 3 independent experiments).

TNFa inhibition with C87 sensitized glioblastoma cells to EGFR inhibition. After C87 (2.5 µM), or TNFa antibody (1 µg/mL), or gefitinib (2 µM) alone, or combination treated U87vIII (A) or LN229vIII (B) cells for 48 hours, cell viability was tested by MTT assay. (C) Apoptotic assay of U87vIII cells with the above-mentioned treatments. In (A), (B) and (C), data were mean ± SEM, n = 3 independent experiments, Student’s t-test, ^*P < 0.05, ^**P < 0.01. (D) Representative morphology of U87vIII cells with the above-mentioned treatments, scale bar, 100 µm. (E) After U87vIII cells were treated with gefitinib combined with C87 or TNFa antibody for 48 h, expression of total Axl, phospho-Axl, total JNK, phospho-JNK, total EGFR and phospho-EGFR were detected by immunoblotting.

C87 failed to work synergistically with TMZ in the cytotoxic assay of glioblastoma cells. (A) In U87vIII cells (left) or LN229vIII cells (right), TMZ treatment didn’t increase the TNFa mRNA expression. (B) 2 µM C87 had no effect on the sensitivity of U87vIII cells (left) or LN229vIII cells (right) to TMZ. In (A) and (B), data were mean ± SEM, n = 3 independent experiments.

Inhibition of Axl and JNK renders glioblastoma cells sensitive to gefitinib. After Axl inhibitor R428(2 µM), or JNK inhibitor SP600125 Add a space (2 µM), or gefitinib (2 µM) alone, or combination treated U87vIII (A) or LN229vIII (B) cells for 48 hours, cell viability was tested by MTT assay. In (A) and (B), data were mean ± SEM, n = 3 independent experiments, Student’s t-test, ^*P < 0.05, ^**P < 0.01.

C87 sensitized mouse xenograft tumors to gefitinib treatment. (A) U87vIII subcutaneous xenograft tumors were once a day treated with C87 (10 mg/kg) intraperitoneally or gefitinib (50 mg/kg) by oral gavage, or combination of the two. (B) LN229vIII subcutaneous xenograft tumors were treated daily with the same doses as in (A). In (A) and (B), tumor volumes were calculated as indicated in the Method. Values are mean ± SD, n = 6 mice for each group, Student’s t-test, ^***P < 0.001 g (gefitinib group vs. gefitinib + C87 group). Inserts were representative tumor photos of gefitinib group or gefitinib + C87 group. (C) U87vIII (top) or LN229vIII (bottom) subcutaneous xenograft tumors of each group were resected, fixed in formalin, and stained for Ki67. Scale bar, 100 µm.