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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

Morphological changes of HT-29 cells observed under an inverted light microscope. a Untreated HT-29 cells, HT-29 cells treated with water extract of brewers’ rice (WBR) at concentrations of b 16 μg/mL, c 32 μg/mL, and d 64 μg/mL. The cells showed typical characteristics of apoptosis, such as nuclear compaction (NC), apoptotic bodies (AB), membrane blebbing (MB), cellular shrinkage (CS), and nuclear fragmentation (NF) (magnification 400×). Scale bars in the figures indicate 50 μm (Source: Tan et al. [19])
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Fig1: Morphological changes of HT-29 cells observed under an inverted light microscope. a Untreated HT-29 cells, HT-29 cells treated with water extract of brewers’ rice (WBR) at concentrations of b 16 μg/mL, c 32 μg/mL, and d 64 μg/mL. The cells showed typical characteristics of apoptosis, such as nuclear compaction (NC), apoptotic bodies (AB), membrane blebbing (MB), cellular shrinkage (CS), and nuclear fragmentation (NF) (magnification 400×). Scale bars in the figures indicate 50 μm (Source: Tan et al. [19])

Mentions: According to published guidelines, any extract that exerts potentially cytotoxic effects should have an IC50 less than 100 μg/mL [18]. We previously reported that WBR and methanol extract of brewers’ rice (MBR) is cytotoxicity towards colorectal cancer (HT-29) cells with IC50 values of 38.33 ± 6.51 μg/mL and 54.00 ± 5.29 μg/mL, respectively [14], as evaluated through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay, whereas a lactate dehydrogenase (LDH) assay showed IC50 values of 21.88 ± 12.43 μg/mL and 34.50 ± 5.92 μg/mL for WBR and MBR, respectively [19]. The results showed that both WBR and MBR are sensitive to HT-29 cells, indicating that WBR and MBR can inhibit the proliferation of HT-29 cells. However, HT-29 cell line was more sensitive to WBR because the median inhibition concentration values were lower than that of MBR, as confirmed by MTT proliferation and LDH assays. The proliferation of HT-29 cells after treated with WBR is shown in Fig. 1. Therefore, we tested whether WBR can affect the Wnt signaling pathway in HT-29 cells.Fig. 1


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)

Morphological changes of HT-29 cells observed under an inverted light microscope. a Untreated HT-29 cells, HT-29 cells treated with water extract of brewers’ rice (WBR) at concentrations of b 16 μg/mL, c 32 μg/mL, and d 64 μg/mL. The cells showed typical characteristics of apoptosis, such as nuclear compaction (NC), apoptotic bodies (AB), membrane blebbing (MB), cellular shrinkage (CS), and nuclear fragmentation (NF) (magnification 400×). Scale bars in the figures indicate 50 μm (Source: Tan et al. [19])
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Morphological changes of HT-29 cells observed under an inverted light microscope. a Untreated HT-29 cells, HT-29 cells treated with water extract of brewers’ rice (WBR) at concentrations of b 16 μg/mL, c 32 μg/mL, and d 64 μg/mL. The cells showed typical characteristics of apoptosis, such as nuclear compaction (NC), apoptotic bodies (AB), membrane blebbing (MB), cellular shrinkage (CS), and nuclear fragmentation (NF) (magnification 400×). Scale bars in the figures indicate 50 μm (Source: Tan et al. [19])
Mentions: According to published guidelines, any extract that exerts potentially cytotoxic effects should have an IC50 less than 100 μg/mL [18]. We previously reported that WBR and methanol extract of brewers’ rice (MBR) is cytotoxicity towards colorectal cancer (HT-29) cells with IC50 values of 38.33 ± 6.51 μg/mL and 54.00 ± 5.29 μg/mL, respectively [14], as evaluated through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay, whereas a lactate dehydrogenase (LDH) assay showed IC50 values of 21.88 ± 12.43 μg/mL and 34.50 ± 5.92 μg/mL for WBR and MBR, respectively [19]. The results showed that both WBR and MBR are sensitive to HT-29 cells, indicating that WBR and MBR can inhibit the proliferation of HT-29 cells. However, HT-29 cell line was more sensitive to WBR because the median inhibition concentration values were lower than that of MBR, as confirmed by MTT proliferation and LDH assays. The proliferation of HT-29 cells after treated with WBR is shown in Fig. 1. Therefore, we tested whether WBR can affect the Wnt signaling pathway in HT-29 cells.Fig. 1

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