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Water extract of brewers' rice induces apoptosis in human colorectal cancer cells via activation of caspase-3 and caspase-8 and downregulates the Wnt/β-catenin downstream signaling pathway in brewers' rice-treated rats with azoxymethane-induced colon carcinogenesis.

Tan BL, Norhaizan ME, Huynh K, Heshu SR, Yeap SK, Hazilawati H, Roselina K - BMC Complement Altern Med (2015)

Bottom Line: Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells.We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and -8 activities compared with the control (P < 0.05).We provide evidence that brewers' rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. tbeeling87@gmail.com.

ABSTRACT

Background: Brewers' rice, is locally known as temukut, is a mixture of broken rice, rice bran, and rice germ. The current study is an extension of our previous work, which demonstrated that water extract of brewers' rice (WBR) induced apoptosis in human colorectal cancer (HT-29) cells. We also identified that brewers' rice was effective in reducing the tumor incidence and multiplicity in azoxymethane (AOM)-injected colon cancer rats. Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells. To further determine whether the in vitro mechanisms of action observed in the HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers' rice in regulating the expressions and key protein markers during colon carcinogenesis in male Sprague-Dawley rats.

Methods: The mRNA levels of several upstream-related genes in the Wnt signaling pathway in HT-29 cells treated with WBR were determined by quantitative real-time PCR analyses. Caspase-3 and -8 were evaluated using a colorimetric assay. Male Sprague-Dawley rats were administered two intraperitoneal injections of AOM in saline (15 mg/kg body weight) over a two-week period and received with 10, 20, and 40% (w/w) brewers' rice. The expressions and protein levels of cyclin D1 and c-myc were evaluated by immunohistochemical staining and western blotting, respectively.

Results: The overall analyses revealed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the casein kinase 1 (CK1) and adenomatous polyposis coli (APC) mRNA levels. We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and -8 activities compared with the control (P < 0.05). In vivo analyses indicated that brewers' rice diminished the β-catenin, cyclin D1, and c-myc protein levels.

Conclusions: We provide evidence that brewers' rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo. We suggest that brewers' rice may be a useful dietary agent for colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

Western blotting of β-catenin, cyclin D1, and c-myc in brewers’ rice treated AOM-induced colon cancer. The protein levels of β-catenin, cyclin D1, and c-myc in normal (Group 1), AOM alone (untreated) (Group 2) compared to groups of rats treated with 10 % (Group 3), 20 % (Group 4), and 40 % (Group 5) (w/w) of brewers’ rice. a Brewers’ rice decreased protein expression of β-catenin, b brewers’ rice reduced protein expression of cyclin D1, and c brewers’ rice decreased protein expression of c-myc in a dose-dependent manner. Each value expressed as mean ± SD (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Administration of 10, 20, and 40 % (w/w) of brewers’ rice significantly reduced β-catenin, cyclin D1, and c-myc protein expression compared to the AOM-alone group (P < 0.05). However, the protein levels of cyclin D1 and c-myc did not significantly differ between 10 % and 20 % (w/w) brewers’ rice (P > 0.05)
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Fig7: Western blotting of β-catenin, cyclin D1, and c-myc in brewers’ rice treated AOM-induced colon cancer. The protein levels of β-catenin, cyclin D1, and c-myc in normal (Group 1), AOM alone (untreated) (Group 2) compared to groups of rats treated with 10 % (Group 3), 20 % (Group 4), and 40 % (Group 5) (w/w) of brewers’ rice. a Brewers’ rice decreased protein expression of β-catenin, b brewers’ rice reduced protein expression of cyclin D1, and c brewers’ rice decreased protein expression of c-myc in a dose-dependent manner. Each value expressed as mean ± SD (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Administration of 10, 20, and 40 % (w/w) of brewers’ rice significantly reduced β-catenin, cyclin D1, and c-myc protein expression compared to the AOM-alone group (P < 0.05). However, the protein levels of cyclin D1 and c-myc did not significantly differ between 10 % and 20 % (w/w) brewers’ rice (P > 0.05)

Mentions: The protein level of β-catenin was diminished accordingly when the concentration of brewers’ rice was increased, indicating that brewers’ rice increased the degradation of β-catenin in a dose-dependent manner (Fig. 7a). Expectedly, we found no β-catenin protein expression in the normal colon tissue. This result may reveal that the β-catenin amount in the normal colon tissue was too low to be detected through western blotting. In the AOM-alone group (G2), a carcinogen was injected, but no treatment with brewers’ rice was administered, and prominent β-catenin protein expression was observed. The overall analysis showed that the colon mucosa of G2, which had the highest values of tumor incidence and tumor multiplicity compared with other groups [16], displayed the highest β-catenin protein expression in the western blotting evaluation compared with the other treatment groups.Fig. 7


Water extract of brewers' rice induces apoptosis in human colorectal cancer cells via activation of caspase-3 and caspase-8 and downregulates the Wnt/β-catenin downstream signaling pathway in brewers' rice-treated rats with azoxymethane-induced colon carcinogenesis.

Tan BL, Norhaizan ME, Huynh K, Heshu SR, Yeap SK, Hazilawati H, Roselina K - BMC Complement Altern Med (2015)

Western blotting of β-catenin, cyclin D1, and c-myc in brewers’ rice treated AOM-induced colon cancer. The protein levels of β-catenin, cyclin D1, and c-myc in normal (Group 1), AOM alone (untreated) (Group 2) compared to groups of rats treated with 10 % (Group 3), 20 % (Group 4), and 40 % (Group 5) (w/w) of brewers’ rice. a Brewers’ rice decreased protein expression of β-catenin, b brewers’ rice reduced protein expression of cyclin D1, and c brewers’ rice decreased protein expression of c-myc in a dose-dependent manner. Each value expressed as mean ± SD (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Administration of 10, 20, and 40 % (w/w) of brewers’ rice significantly reduced β-catenin, cyclin D1, and c-myc protein expression compared to the AOM-alone group (P < 0.05). However, the protein levels of cyclin D1 and c-myc did not significantly differ between 10 % and 20 % (w/w) brewers’ rice (P > 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: Western blotting of β-catenin, cyclin D1, and c-myc in brewers’ rice treated AOM-induced colon cancer. The protein levels of β-catenin, cyclin D1, and c-myc in normal (Group 1), AOM alone (untreated) (Group 2) compared to groups of rats treated with 10 % (Group 3), 20 % (Group 4), and 40 % (Group 5) (w/w) of brewers’ rice. a Brewers’ rice decreased protein expression of β-catenin, b brewers’ rice reduced protein expression of cyclin D1, and c brewers’ rice decreased protein expression of c-myc in a dose-dependent manner. Each value expressed as mean ± SD (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Administration of 10, 20, and 40 % (w/w) of brewers’ rice significantly reduced β-catenin, cyclin D1, and c-myc protein expression compared to the AOM-alone group (P < 0.05). However, the protein levels of cyclin D1 and c-myc did not significantly differ between 10 % and 20 % (w/w) brewers’ rice (P > 0.05)
Mentions: The protein level of β-catenin was diminished accordingly when the concentration of brewers’ rice was increased, indicating that brewers’ rice increased the degradation of β-catenin in a dose-dependent manner (Fig. 7a). Expectedly, we found no β-catenin protein expression in the normal colon tissue. This result may reveal that the β-catenin amount in the normal colon tissue was too low to be detected through western blotting. In the AOM-alone group (G2), a carcinogen was injected, but no treatment with brewers’ rice was administered, and prominent β-catenin protein expression was observed. The overall analysis showed that the colon mucosa of G2, which had the highest values of tumor incidence and tumor multiplicity compared with other groups [16], displayed the highest β-catenin protein expression in the western blotting evaluation compared with the other treatment groups.Fig. 7

Bottom Line: Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells.We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and -8 activities compared with the control (P < 0.05).We provide evidence that brewers' rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. tbeeling87@gmail.com.

ABSTRACT

Background: Brewers' rice, is locally known as temukut, is a mixture of broken rice, rice bran, and rice germ. The current study is an extension of our previous work, which demonstrated that water extract of brewers' rice (WBR) induced apoptosis in human colorectal cancer (HT-29) cells. We also identified that brewers' rice was effective in reducing the tumor incidence and multiplicity in azoxymethane (AOM)-injected colon cancer rats. Our present study was designed to identify whether WBR confers an inhibitory effect via the regulation of upstream components in the Wnt signaling pathway in HT-29 cells. To further determine whether the in vitro mechanisms of action observed in the HT-29 cells inhibit the downstream signaling target of the Wnt/β-catenin pathway, we evaluated the mechanistic action of brewers' rice in regulating the expressions and key protein markers during colon carcinogenesis in male Sprague-Dawley rats.

Methods: The mRNA levels of several upstream-related genes in the Wnt signaling pathway in HT-29 cells treated with WBR were determined by quantitative real-time PCR analyses. Caspase-3 and -8 were evaluated using a colorimetric assay. Male Sprague-Dawley rats were administered two intraperitoneal injections of AOM in saline (15 mg/kg body weight) over a two-week period and received with 10, 20, and 40% (w/w) brewers' rice. The expressions and protein levels of cyclin D1 and c-myc were evaluated by immunohistochemical staining and western blotting, respectively.

Results: The overall analyses revealed that the treatment of HT-29 cells with WBR inhibited Wnt signaling activity through upregulation of the casein kinase 1 (CK1) and adenomatous polyposis coli (APC) mRNA levels. We discovered that the treatment of HT-29 cells with WBR resulted in the induction of apoptosis by the significant activation of caspase-3 and -8 activities compared with the control (P < 0.05). In vivo analyses indicated that brewers' rice diminished the β-catenin, cyclin D1, and c-myc protein levels.

Conclusions: We provide evidence that brewers' rice can induce apoptosis and inhibit the proliferation of HT-29 cells through regulation of caspase-dependent pathways and inhibit the Wnt/β-catenin downstream signaling pathway in vivo. We suggest that brewers' rice may be a useful dietary agent for colorectal cancer.

No MeSH data available.


Related in: MedlinePlus