Article Figures & Data
Figures
- Fig. 1.
Immunohistochemical analysis of NF-κB subunits in patient matched HNSCC and noncancerous epithelium biopsies. A, SCC of tonsil (bottom; magnification, 400×) compared with epithelium from opposite tonsil (top; magnification, 400×). B, SCC of tongue (bottom; magnification, 400×) compared with biopsy from adjacent lingual epithelium (top; magnification, 400×). Both SCC biopsies show diffuse positive nuclear staining of variable intensity of all five NF-κB subunits. NF-κB subunit nuclear staining limited to the basal layers is shown in the matched noncancerous epithelia, with nuclear staining reduced or lost in the suprabasal layers. p-RELA, phosphorylated RELA (Ser536); Isotype, rabbit IgG isotype-negative control.
- Fig. 2.
Immunohistochemistry analysis of pretreatment and 24-h posttreatment biopsies from patients 1 and 4 after 0.6 mg/m2 bortezomib. Patient 1 (A) shows decreased staining from pretreatment (top; magnification, 1,000×) to posttreatment (bottom; magnification, 1,000×) in phosphorylated RELA, c-REL, p50, p52, and Ki67, as well as increased TUNEL staining, whereas RELB seems to be unaffected. Conversely, patient 4 (B) shows very weak pretreatment nuclear phosphorylated RELA and no change after bortezomib therapy in NF-κB subunit nuclear localization or TUNEL staining, but did show decreased Ki67. In both patients, activated ERK1/2 and STAT3 are unaffected by bortezomib therapy. p-ERK1/2, phosphorylated ERK (Thr202/Tyr204); p-STAT3, phosphorylated STAT3 (Tyr705); pre-tx, pretreatment; post-tx, posttreatment.
- Fig. 3.
Panel of histograms summarizing the pretreatment and posttreatment apoptotic indices, proliferation indices, and histoscores for four patients with evaluable biopsies. In patients 1 to 3 (A-C), decreased activated nuclear RELA correlated with increased apoptosis measured via TUNEL assay. Patient 4 (D) showed no decrease in activated RELA and no increase in TUNEL. Change in nuclear localization of total c-REL, RELB, p50, and p52 was variable among the four patients. Decreased proliferation, measured via Ki67 staining, was consistently observed after bortezomib therapy. Nuclear staining of activated ERK1/2 and STAT3 was unaffected by bortezomib in four of four patients. Staining scores shown as a mean of three high power fields. *, statistically significant differences between pretreatment and posttreatment staining scores (P < 0.05, Student's t test).
- Fig. 4.
Baseline NF-κB subunit localization and DNA binding activity of HNSCC cell lines. A, Western blot analysis of cytoplasmic and nuclear fractions reveals variable by positive nuclear localization of total RELA, RELB, c-REL, p50, and p52. Whereas UMSCC-11B shows the greatest relative quantity of nuclear RELA, RELB, p50, and p52, UMSCC-9 shows the greatest relative amount of c-REL. Greater relative amounts of the precursor proteins p105 and p100 were found in the cytoplasm compared with the nucleus. B, compared with negative and wild-type oligonucleotide controls, all three cell lines show significant constitutive DNA binding activity of RELA, p50, RELB, and p52, whereas only UMSCC-9 shows weak but significant constitutive c-REL DNA binding. *, significant (P < 0.05) increase in DNA binding above the negative control; †, significant (P > 0.05) increase in DNA binding above the wild-type competition oligonucleotide.
- Fig. 5.
Cytotoxicity and altered DNA binding activity after bortezomib treatment in HNSCC cell lines. A, MTT assays reveal variable cytotoxicity to low dose bortezomib. High dose (10−7 mol/L) bortezomib caused significant cell death universally by day 1, low dose (10−9 mol/L) caused significant cell death in UMSCC-11B by day 5 only, and intermediate dose (10−8 mol/L) caused significant cytotoxicity by day 1 in UMSCC-9 and UMSCC-11B and by day 3 in UMSCC-11A. *, first time point at which 10−9 mol/L bortezomib shows significant (P < 0.05) cytotoxicity compared with control (untreated) cells; **, first time point at which 10−8 mol/L bortezomib shows significant cytotoxicity; †, first time point at which 10−7 mol/L bortezomib shows significant (P < 0.05) cytotoxicity. B and C, inhibition of TNF-α inducible RELA and p50 DNA binding, respectively. There seems to be no early (4 h) difference in inhibition of inducible RELA or p50 DNA binding by 10−9 or 10−8 mol/L bortezomib. However, as inhibition by 10−8 mol/L increased with time, inhibition by 10−9 mol/L remains constant or decreases with time creating significant differences in inhibition by the 12-h or 24-h time points. ‡, significant (P < 0.05) difference in inhibition of inducible activity.
Tables
- Table 1.
Patient tumor and treatment characteristics and clinical outcomes
Patient Tumor histology Tumor location Therapy before recurrence Bortezomib dose Bortezomib schedule* Posttreatment biopsy† Local control on treatment; by 3 mo‡ 1 Recurrent SCC Floor of mouth and anterior triangle neck S, C, R 0.6 mg/m2 Twice weekly × 7 wk without break Yes 50% tumor reduction; PD below field at 4 wk 2 Recurrent SCC Base of tongue and anterior triangle neck S, C, R 0.6 mg/m2 Twice weekly × 7 wk without break Yes PD below field at 2 wk on treatment 3 Recurrent SCC Left base of tongue and vallecula S, R 0.6 mg/m2 Twice weekly × 7 wk with 2 wk break Yes Stable disease on treatment; PD in field at 3 mo 4 Recurrent SCC Anterior neck and parapharyngeal space S, C, R 0.6 mg/m2 Twice weekly × 7 wk without break Yes Stable disease on treatment PD out of field at 1 wk 5 Recurrent SCC Anterior neck with tracheal extension S, R 0.6 mg/m2 Twice weekly × 7 wk without break No 90% tumor reduction PD in field at 5 mo 6 Recurrent SCC Base of tongue S, R 0.6 mg/m2 Twice weekly × 7 wk with 2 wk break No 30% tumor reduction PD 3 mo NOTE: S, surgery; C, chemotherapy; R, radiation; PD, progressive disease.
↵* Bortezomib given concurrently with fractionated radiotherapy (to 50-70 Gy) given daily (Monday to Friday).
↵† Timing of all posttreatment biopsies is 24 h after initial treatment before the start of radiotherapy.
↵‡ Based upon RECIST criteria, stable disease is defined as <30% tumor reduction and partial response is defined as >30% tumor reduction.
Supplementary Data, Allen, et al.
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Volume 14, Issue 13
