Synthetic curcumin analog EF31 inhibits the growth of head and neck squamous cell carcinoma xenografts

Abstract

Objectives are to examine the efficacy, pharmacokinetics, and toxicology of a synthetic curcumin analog EF31 in head and neck squamous cell carcinoma. The synthesis of EF31 was described for the first time. Solubility of EF24 and EF31 was compared using nephelometric analysis. Human head and neck squamous cell carcinoma Tu212 xenograft tumors were established in athymic nude mice and treated with EF31 i.p. once daily five days a week for about 5-6 weeks. The long term effect of EF31 on the NF-κB signaling system in the tumors was examined by Western blot analysis. EF31 at 25 mg kg(-1), i.p. inhibited tumor growth almost completely. Solubilities of EF24 and EF31 are <10 and 13 μg mL(-1) or <32 and 47 μM, respectively. The serum chemistry profiles of treated mice were within the limits of normal, they revealed a linear increase of C(max). EF31 decreased the level of phosphorylation of NF-κB p65. In conclusion, the novel synthetic curcumin analog EF31 is efficacious in inhibiting the growth of Tu212 xenograft tumors and may be useful for treating head and neck squamous cell carcinoma. The long term EF31 treatment inhibited NF-κB p65 phosphorylation in xenografts, implicating downregulation of cancer promoting transcription factors such as angiogenesis and metastasis.

Fig. 1. Cytotoxic activity and solubility of EF24 and EF31 against Tu212 SCC cells in vitro

Chemical structures of curcumin, EF24, EF31 are shown. Molecular weight of curcumin, EF24 and EF31 are 368.38, 311.11 g/mol, respectively (Fig. 1A). Tu212 cells (10⁴ cells/0.2 ml/well) in a 96-well plate were incubated with varying doses of EF24, EF31 (0 – 20 μM) in triplicate for 48 hours. Viable cells were measured by Neutral Red Dye assay. IC₅₀ values of EF24, EF31 were 8, 7μM, respectively (Fig. 1B). The aqueous solubilities of EF24, EF31 were determined by nephelometry to be < 10 μg/mL (32 μM), 13 μg/mL (47 μM), respectively. Solubilities of EF24, EF31 are shown as the concentrations at which the solute began to precipitate out of solution and expressed as counts of refractive nephelometry units (RNU) (Fig. 1C).

Fig. 2. Efficacy of EF31 in 0.5% carboxymethyl cellulose sodium (CMC) and 10% DMSO by i.p. in Tu212 SCC xenografts in female athymic nude mice

Tu212 cells (2 × 10⁶/0.1 ml medium) were inoculated subcutaneously (s.c.). EF31 (12.5 mg/kg and 25 mg/kg) or vehicle were administered intraperitoneally (i.p.) to mice (n = 10/group) daily for 5 days a week (Monday through Friday) for 5 weeks. Treatment was started when tumors grew to approximately 0.2–0.3 cm in diameter. (A) Tumor size. Asterisks indicate significant difference from the control (p < 0.05–0.01). (B) EF31 at 25 mg/kg reduced body weight temporarily by ~10% on average but animals regained body weight as treatment continued.

Fig. 3. Pharmacokinetic study of EF31 administered by i.p.

(A, B) After i.p. administration of EF31 at 25 mg/kg in a DMSO/0.5% CMC (10%/90%) formulation, peak mouse plasma concentrations were reached at 0.5 hours post-dose (Tmax) with an average concentration (Cmax) of 2127 ng/mL. The terminal elimination half-life (t½) averaged 2.4 hours, with an average AUC(0-∞) of 7769 hr × ng/mL. Both Cmax and Tmax were dose proportional following i.p. administration of EF31 to mice at 12.5 and 25 mg/kg.

Fig. 4. Changes in the NF-κB pathways in tumors treated with EF31 (Western blots)

Tumor bearing animals were treated with EF31 0, 12.5 mg/kg and 25 mg/kg i.p. daily for 5 days a week for 5 weeks as shown in Fig. 2. Tumor extracts were analyzed by Western blotting using various antibodies for NF-κB pathways.

Fig. 5. Hemotoxylin and Eosin staining (×100) and corresponding immunohistochemistry patterns of xenografts mouse tumor tissues

(A) H & E staining, tumor bearing animals were treated with EF31 0, 12.5 mg/kg and 25 mg/kg i.p. daily for 5 days a week for 5 weeks. (B) IKKβ and phospho-IKKβ staining of mice tumor tissues serial sections.(C) IkBα and phospho- IkBα staining of mice tumor tissues serial sections.(D) NF-κB p65 and NF-κB phospho-p65 staining of mice tumor tissues serial sections. The scale bars indicate 100 μm.

Fig. 5. Hemotoxylin and Eosin staining (×100) and corresponding immunohistochemistry patterns of xenografts mouse tumor tissues

(A) H & E staining, tumor bearing animals were treated with EF31 0, 12.5 mg/kg and 25 mg/kg i.p. daily for 5 days a week for 5 weeks. (B) IKKβ and phospho-IKKβ staining of mice tumor tissues serial sections.(C) IkBα and phospho- IkBα staining of mice tumor tissues serial sections.(D) NF-κB p65 and NF-κB phospho-p65 staining of mice tumor tissues serial sections. The scale bars indicate 100 μm.

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