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Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer

View Article: PubMed Central - PubMed

ABSTRACT

Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2.

No MeSH data available.


Related in: MedlinePlus

Autophagy deficiency was associated with the sensitivity of CRC cells to GW8510. (a) The protein expressions in HCT116, LoVo, HCT15, DLD-1, and HT-29 cells were analyzed by western blots. The ratio of LC3-II to LC3-I or GAPDH was quantified. (b) HCT116 cells were treated the different doses of GW8510 for 72 h in the absence or presence of 10 nM bafilomycin. The cell viability was determined by an MTT assay. (c) HCT116 cells were treated with different doses of rapamycin (Rapa) for 24 h. The protein expressions were analyzed by western blots. (d) ATG7-wild-type (ATG7-WT) and ATG7-knockout (ATG7-KO) DLD-1 cells were treated with different doses of GW8510 for 72 h. The cell viability was determined by an MTT assay.
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fig5: Autophagy deficiency was associated with the sensitivity of CRC cells to GW8510. (a) The protein expressions in HCT116, LoVo, HCT15, DLD-1, and HT-29 cells were analyzed by western blots. The ratio of LC3-II to LC3-I or GAPDH was quantified. (b) HCT116 cells were treated the different doses of GW8510 for 72 h in the absence or presence of 10 nM bafilomycin. The cell viability was determined by an MTT assay. (c) HCT116 cells were treated with different doses of rapamycin (Rapa) for 24 h. The protein expressions were analyzed by western blots. (d) ATG7-wild-type (ATG7-WT) and ATG7-knockout (ATG7-KO) DLD-1 cells were treated with different doses of GW8510 for 72 h. The cell viability was determined by an MTT assay.

Mentions: To further investigate the mechanism responsible for the differential sensitivities of CRC cells to GW8510, the relative RRM2 expression levels in these cells were compared by western blot analysis. As shown in Figure 5a, both HCT15 and DLD-1 showed the highest level of RRM2. However, overexpression of RRM2 could not fully explain their differential sensitivities to GW8510 because of the relatively low RRM2 expression in HT-29 cells (Figure 5a). As GW8510 induced autophagic cell death of CRC cells, we hypothesized that, in addition to the level of RRM2, the endogenous abilities of CRC cells to undergo autophagy also determined the cell fate. Thus, the expression of LC3 and p62 was examined by western blot analysis to estimate the overall autophagic flux in cells.19 As shown in Figure 5a, GW8510-sensitive cells (HCT116 and LoVo) had higher LC3-II accumulation and lower p62 expression than GW8510-resistant cells (HCT15, DLD-1, and HT-29). Although p62 expression in LoVo cells were relatively high among these cells, the accelerated autophagic flux (higher LC3-II/LC3-I ratio) might be able to compensate for high p62 expression. To confirm the role of autophagy in the sensitivity of GW8510, HCT116 cells were treated with bafilomycin A1 to block autophagy flux. Bafilomycin A1 is a vacuolar-type H+-ATPase inhibitor that blocks autophagosome-lysosome fusion.19 Treatment with bafilomycin attenuated the anticancer activity of GW8510 (Figure 5b). Furthermore, autophagy-deficient ATG7-knockout (ATG7-KO) DLD-1 cells were used. The impairment of autophagy in ATG7-KO cells was examined by treating with an autophagy inducer, rapamycin. As shown in Figure 5c, rapamycin could trigger autophagy (LC3-II accumulation and p62 degradation) in ATG7-wild-type (ATG7-WT) DLD-1 cells, but not in ATG7-KO cells. In addition, p62 accumulation was found in ATG7-KO cells (Figure 5c). Consistent with the effect by bafilomycin (Figure 5b), ATG7-KO cells were resistant to GW8510 treatment (Figure 5d). Taken together, both RRM2 overexpression and impaired autophagic flux in CRC cells contributes the resistance of GW8510.


Repositioning of a cyclin-dependent kinase inhibitor GW8510 as a ribonucleotide reductase M2 inhibitor to treat human colorectal cancer
Autophagy deficiency was associated with the sensitivity of CRC cells to GW8510. (a) The protein expressions in HCT116, LoVo, HCT15, DLD-1, and HT-29 cells were analyzed by western blots. The ratio of LC3-II to LC3-I or GAPDH was quantified. (b) HCT116 cells were treated the different doses of GW8510 for 72 h in the absence or presence of 10 nM bafilomycin. The cell viability was determined by an MTT assay. (c) HCT116 cells were treated with different doses of rapamycin (Rapa) for 24 h. The protein expressions were analyzed by western blots. (d) ATG7-wild-type (ATG7-WT) and ATG7-knockout (ATG7-KO) DLD-1 cells were treated with different doses of GW8510 for 72 h. The cell viability was determined by an MTT assay.
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fig5: Autophagy deficiency was associated with the sensitivity of CRC cells to GW8510. (a) The protein expressions in HCT116, LoVo, HCT15, DLD-1, and HT-29 cells were analyzed by western blots. The ratio of LC3-II to LC3-I or GAPDH was quantified. (b) HCT116 cells were treated the different doses of GW8510 for 72 h in the absence or presence of 10 nM bafilomycin. The cell viability was determined by an MTT assay. (c) HCT116 cells were treated with different doses of rapamycin (Rapa) for 24 h. The protein expressions were analyzed by western blots. (d) ATG7-wild-type (ATG7-WT) and ATG7-knockout (ATG7-KO) DLD-1 cells were treated with different doses of GW8510 for 72 h. The cell viability was determined by an MTT assay.
Mentions: To further investigate the mechanism responsible for the differential sensitivities of CRC cells to GW8510, the relative RRM2 expression levels in these cells were compared by western blot analysis. As shown in Figure 5a, both HCT15 and DLD-1 showed the highest level of RRM2. However, overexpression of RRM2 could not fully explain their differential sensitivities to GW8510 because of the relatively low RRM2 expression in HT-29 cells (Figure 5a). As GW8510 induced autophagic cell death of CRC cells, we hypothesized that, in addition to the level of RRM2, the endogenous abilities of CRC cells to undergo autophagy also determined the cell fate. Thus, the expression of LC3 and p62 was examined by western blot analysis to estimate the overall autophagic flux in cells.19 As shown in Figure 5a, GW8510-sensitive cells (HCT116 and LoVo) had higher LC3-II accumulation and lower p62 expression than GW8510-resistant cells (HCT15, DLD-1, and HT-29). Although p62 expression in LoVo cells were relatively high among these cells, the accelerated autophagic flux (higher LC3-II/LC3-I ratio) might be able to compensate for high p62 expression. To confirm the role of autophagy in the sensitivity of GW8510, HCT116 cells were treated with bafilomycin A1 to block autophagy flux. Bafilomycin A1 is a vacuolar-type H+-ATPase inhibitor that blocks autophagosome-lysosome fusion.19 Treatment with bafilomycin attenuated the anticancer activity of GW8510 (Figure 5b). Furthermore, autophagy-deficient ATG7-knockout (ATG7-KO) DLD-1 cells were used. The impairment of autophagy in ATG7-KO cells was examined by treating with an autophagy inducer, rapamycin. As shown in Figure 5c, rapamycin could trigger autophagy (LC3-II accumulation and p62 degradation) in ATG7-wild-type (ATG7-WT) DLD-1 cells, but not in ATG7-KO cells. In addition, p62 accumulation was found in ATG7-KO cells (Figure 5c). Consistent with the effect by bafilomycin (Figure 5b), ATG7-KO cells were resistant to GW8510 treatment (Figure 5d). Taken together, both RRM2 overexpression and impaired autophagic flux in CRC cells contributes the resistance of GW8510.

View Article: PubMed Central - PubMed

ABSTRACT

Colorectal cancer (CRC) is the second leading cause of cancer-related death in males and females in the world. It is of immediate importance to develop novel therapeutics. Human ribonucleotide reductase (RRM1/RRM2) has an essential role in converting ribonucleoside diphosphate to 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. RRM2 is a prognostic biomarker and predicts poor survival of CRC. In addition, increased RRM2 activity is associated with malignant transformation and tumor cell growth. Bioinformatics analyses show that RRM2 was overexpressed in CRC and might be an attractive target for treating CRC. Therefore, we attempted to search novel RRM2 inhibitors by using a gene expression signature-based approach, connectivity MAP (CMAP). The result predicted GW8510, a cyclin-dependent kinase inhibitor, as a potential RRM2 inhibitor. Western blot analysis indicated that GW8510 inhibited RRM2 expression through promoting its proteasomal degradation. In addition, GW8510 induced autophagic cell death. In addition, the sensitivities of CRC cells to GW8510 were associated with the levels of RRM2 and endogenous autophagic flux. Taken together, our study indicates that GW8510 could be a potential anti-CRC agent through targeting RRM2.

No MeSH data available.


Related in: MedlinePlus