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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

Caspase-3 and −8 activities in HT-29 cells treated with WBR (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Both caspase-3 and −8 activities in the groups treated with WBR were significantly increased compared with the control (P < 0.05)
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Fig4: Caspase-3 and −8 activities in HT-29 cells treated with WBR (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Both caspase-3 and −8 activities in the groups treated with WBR were significantly increased compared with the control (P < 0.05)

Mentions: As shown in Fig. 4, WBR clearly increases the caspase-3 and −8 activities in a dose-dependent manner. The caspase-3 activity after treatment with 16, 32, and 64 μg/mL WBR was significantly increased compared with the control (P < 0.05). In addition to the upregulation of caspase-3, the caspase-8 activity in HT-29 cells was also significantly increased after treatment with WBR for 72 h (P < 0.05). Therefore, the findings may suggest that an increase in caspase-8 activity result in the stimulation of the downstream apoptotic executioner caspase-3, which subsequently activates the molecular cascade of apoptosis in HT-29 cells. Taken together, our data indicate that the caspase-3 and −8 activities are induced by WBR in a dose-dependent manner. WBR markedly increase in apoptosis, which was accompanied by the activation of caspase-3 and −8 activities. Collectively, these results demonstrated that the activation of caspase activities in HT-29 cells after treatment with WBR increments initiator caspase-8 and executioner caspase-3. Our data presented in this study demonstrated that WBR inhibits the proliferation of colon cancer in vitro, as reported in our earlier study [19], leading to programmed cell death, which was confirmed to be through the regulation of caspase-dependent pathways. To further verify whether the in vitro mechanisms of action observed in HT-29 cells could suppress the downstream signaling target of the Wnt/β-catenin pathway, we investigated whether brewers’ rice can reduce the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats using immunohistochemical and western blot evaluations. Thus, the levels of the downstream signaling targets cyclin D1 and c-myc in response to brewers’ rice were further evaluated in the colons of rats injected with AOM.Fig. 4


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)

Caspase-3 and −8 activities in HT-29 cells treated with WBR (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Both caspase-3 and −8 activities in the groups treated with WBR were significantly increased compared with the control (P < 0.05)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487214&req=5

Fig4: Caspase-3 and −8 activities in HT-29 cells treated with WBR (n = 3). Value with different superscript letter indicates significant difference between groups by Tukey’s test (P < 0.05). Both caspase-3 and −8 activities in the groups treated with WBR were significantly increased compared with the control (P < 0.05)
Mentions: As shown in Fig. 4, WBR clearly increases the caspase-3 and −8 activities in a dose-dependent manner. The caspase-3 activity after treatment with 16, 32, and 64 μg/mL WBR was significantly increased compared with the control (P < 0.05). In addition to the upregulation of caspase-3, the caspase-8 activity in HT-29 cells was also significantly increased after treatment with WBR for 72 h (P < 0.05). Therefore, the findings may suggest that an increase in caspase-8 activity result in the stimulation of the downstream apoptotic executioner caspase-3, which subsequently activates the molecular cascade of apoptosis in HT-29 cells. Taken together, our data indicate that the caspase-3 and −8 activities are induced by WBR in a dose-dependent manner. WBR markedly increase in apoptosis, which was accompanied by the activation of caspase-3 and −8 activities. Collectively, these results demonstrated that the activation of caspase activities in HT-29 cells after treatment with WBR increments initiator caspase-8 and executioner caspase-3. Our data presented in this study demonstrated that WBR inhibits the proliferation of colon cancer in vitro, as reported in our earlier study [19], leading to programmed cell death, which was confirmed to be through the regulation of caspase-dependent pathways. To further verify whether the in vitro mechanisms of action observed in HT-29 cells could suppress the downstream signaling target of the Wnt/β-catenin pathway, we investigated whether brewers’ rice can reduce the expressions and key protein markers during colon carcinogenesis in male Sprague–Dawley rats using immunohistochemical and western blot evaluations. Thus, the levels of the downstream signaling targets cyclin D1 and c-myc in response to brewers’ rice were further evaluated in the colons of rats injected with AOM.Fig. 4

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