Figure 6.

Expression and methylation status of O ⁶‐methylguanine‐DNA methyltransferase (MGMT) and mismatch repair (MMR)‐related molecules in post‐treatment recurred U87MG.∆EGFR escaper tumor cells (U87MG.∆EGFR escaper) and their treatment sensitivity in vivo. Nude mice bearing U87MG.∆EGFR xenografts were treated with either vehicles (Vehicles or V), nimotuzumab alone (Nimo or N), TMZ alone (TMZ or T), or combination with nimotuzumab and TMZ (Nimo+TMZ or NT) as in Fig. 3, and the tumors which had regrown (escapers) to a full size after the treatments were removed. Regrown tumors were put back to cell culture to re‐establish escaper cell lines. A. Western blot analysis of lysates from regrown xenografts and re‐established cultured cells (Culture) showing reduced expression of MSH6 and MLH1 by NT treatment. β‐actin blot was used as an internal control. Relative expression level of MMR proteins is shown below each lane, calculated as a ratio of that of control and standardized with actin expression. For MGMT, the highest expression in NT treatment is used as a control. Molecular sizes were shown at the right margin of the panels. B. Methylation‐specific PCR (MSP) for the MGMT promoter region showing the totally methylated pattern in all escaper xenografts. The presence of a PCR product in the U Lane signifies the presence of unmethylated MGMT promoter (undetectable in all samples), whereas a PCR product in the M Lane indicates the presence of methylated promoter. Normal peripheral blood leukocyte DNA (NC) was negative for methylation, whereas human genomic DNA with enzymatic methylation (PC) was used as a positive control for methylation. C, D. Abrogation of sensitivity to nimotuzumab/TMZ combination treatment in NT‐escaper intracerebral tumors. BALB/CA mice bearing intracerebral xenografts derived from U87MG.∆EGFR‐NT‐escaper (C), or U87MG.∆EGFR parental cells (D) were treated with vehicles (Vehicles or V), nimotuzumab alone (Nimo or N), TMZ alone (TMZ or T), or combination with nimotuzumab and TMZ (Nimo+TMZ or NT) as in Fig. 3. Survival curves for U87MG.∆EGFR parental intracerebral tumors were shown as a control for the treatment (P = 0.007, log‐rank test).