Randomized phase II trial of lyophilized strawberries in patients with dysplastic precancerous lesions of the esophagus

Abstract

Dysplasia is a histologic precursor of esophageal squamous cell carcinoma (SCC). We previously showed that dietary freeze-dried, or lyophilized, strawberry powder inhibits N-nitrosomethylbenzylamine-induced SCC in the rat esophagus. On the basis of this observation, we conducted a randomized (noncomparative) phase II trial in China to investigate the effects of two doses of freeze-dried strawberries in patients with esophageal dysplastic lesions in a high-risk area for esophageal cancer. We randomly assigned 75 patients identified by endoscopy to have dysplastic esophageal premalignant lesions to receive freeze-dried strawberry powder at either 30 g/d (37 patients) or 60 g/d (38 patients) for six months; the powder was mixed with water and drunk. After six months, we assessed the changes in histologic grade of these lesions (primary endpoint) in a blinded fashion. The dose of 30 g/d, did not significantly affect histology or any other measured parameter. The dose of 60 g/d, however, reduced the histologic grade of dysplastic premalignant lesions in 29 (80.6%) of the 36 patients at this dose who were evaluated for histology (P < 0.0001). The strawberry powder was well tolerated, with no toxic effects or serious adverse events. Strawberries (60 g/d) also reduced protein expression levels of inducible nitric oxide synthase (iNOS) by 79.5% (P < 0.001), cyclooxygenase-2 (COX-2) by 62.9% (P < 0.001), phospho-nuclear factor kappa B (NFκB)-p65 (pNFκB-p65) by 62.6% (P < 0.001), and phospho-S6 (pS6) by 73.2% (P < 0.001). Freeze-dried strawberries (60 g/d) also significantly inhibited the Ki-67 labeling index by 37.9% (P = 0.023). Our present results indicate the potential of freeze-dried strawberry powder for preventing human esophageal cancer, supporting further clinical testing of this natural agent in this setting.

Figure 1

Trial flow diagram.

Figure 2

Histologic grades of esophageal tissues collected from study participants. A, normal: the epithelium is nonkeratinizing and stratified. B, hyperplasia: there is an increase in the number of basal cells, but no atypia. C and D, dysplasia: there is an increase in the number of basal cells and some of them show nuclear overlapping and hyperchromasia. Mild dysplasia (C)—basal cells showing loss of polarity, composing less than 25% of the total thickness of the epithelium, and covered by thick squamous cell layer, while moderate dysplasia (D)—basal cells showing loss of polarity, composing closer to 50% of the total thickness of the epithelium, and with a reduced squamous layer (magnification ×200).

Figure 3

Representative endoscopic images of esophagus before (A1, 2 and B1, 2) and after (A3, 4 and B3, 4) strawberry treatment. A1, 3 and B1, 3, endoscopic images of esophagus before iodine staining. A flat lesion with white patch (white arrows indicated) can be seen in A1, B1, and B3, although it is not clear. A2, B2, and B4, endoscopic images of the same lesion after iodine staining (white arrows indicated). Well-demarcated unstained mucosal areas are clearly observed. The histopathology reports showed that patient A had mild dysplasia (A1 and 2) before strawberry treatment and these lesions regressed to normal esophagus (A3 and 4) after strawberry treatment. Normal mucosa is smooth or mildly wrinkled and shows a diffuse change to brown with iodine staining (A3 and 4). Patient B had moderate dysplasia (B1 and 2) before treatment and mild dysplasia (B3 and 4) after treatment.

Figure 4

Effect of freeze-dried strawberries in the protein expression of iNOS, COX-2, p-NFκB-p65, and pS6. The indicated proteins were detected by Western blot analyses. Representative blots are shown; similar results were obtained from triplicate experiments. The values are relative densitometric intensity expressed as mean; bars, ± SE. ^**, P < 0.001 as determined by Student t test when compared with the expression level before strawberry treatment.

Figure 5

Effect of freeze-dried strawberries on cell proliferation (A). Strawberries significantly reduced Ki-67 expression after 6-month treatment (B, b) when compared with before treatment (B, a; magnification ×200).

Figure 6

Possible mechanisms for the inhibition of esophageal precancerous progression by strawberries.