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Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

Bottom Line: Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion.Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion.In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.

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HCC stem cells are resistant to conventional chemotherapy. (A, C) After serum starvation of cultured cells overnight, sorafenib and doxorubicin were added at various concentrations (10−3–10−10 M) and cell viability was assessed after 72 hrs. IC50 graph of high-content image analysis of human hepatocytes and HCC stem cells treated by sorafenib and doxorubicin was illustrated. Quantitative data of MTS assay in the sorafenib treated cells were calculated for IC50 with XLfit software, which is marked in the middle of the box. (B, D) IC50 of sorafenib and doxorubicin in hepato-cytes and HCC stem cells is illustrated in bar graph. HCC stem cells under either self-renewal (HSC-SR) or differentiation (HSC-DF) are more resistance to sorafenib and doxorubicin than HepG2 cells and normal liver stem cells. IC50 results are expressed as the mean ± S.E. of eight different experiments. (E) Groups of five SCID Berge mice were selected 8–10 weeks old per treatment group. The human HSC-SRs (1000) as well as parental HCC cells (5 × 106) were injected subcutaneously and allowed to engraft for 10 days. At day 10 the tumour was visible, and then the doxorubicin treatment was started and the mice received the desired concentration of the drug intraper-oteneal three times per week for 14 days. At the end of the 14 days the tumour was dissociated into single cell suspension and assayed with Almar Blue to determine percent inhibition of the drug. (F, G) Differential expression of CD133 and let-7a in vivo following treatment with doxorubicin by real-time PCR is shown. Data represent percentage change in expression in doxo-cibicin-treated tumours compared with controls. *P < 0.05 relative to controls.
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fig04: HCC stem cells are resistant to conventional chemotherapy. (A, C) After serum starvation of cultured cells overnight, sorafenib and doxorubicin were added at various concentrations (10−3–10−10 M) and cell viability was assessed after 72 hrs. IC50 graph of high-content image analysis of human hepatocytes and HCC stem cells treated by sorafenib and doxorubicin was illustrated. Quantitative data of MTS assay in the sorafenib treated cells were calculated for IC50 with XLfit software, which is marked in the middle of the box. (B, D) IC50 of sorafenib and doxorubicin in hepato-cytes and HCC stem cells is illustrated in bar graph. HCC stem cells under either self-renewal (HSC-SR) or differentiation (HSC-DF) are more resistance to sorafenib and doxorubicin than HepG2 cells and normal liver stem cells. IC50 results are expressed as the mean ± S.E. of eight different experiments. (E) Groups of five SCID Berge mice were selected 8–10 weeks old per treatment group. The human HSC-SRs (1000) as well as parental HCC cells (5 × 106) were injected subcutaneously and allowed to engraft for 10 days. At day 10 the tumour was visible, and then the doxorubicin treatment was started and the mice received the desired concentration of the drug intraper-oteneal three times per week for 14 days. At the end of the 14 days the tumour was dissociated into single cell suspension and assayed with Almar Blue to determine percent inhibition of the drug. (F, G) Differential expression of CD133 and let-7a in vivo following treatment with doxorubicin by real-time PCR is shown. Data represent percentage change in expression in doxo-cibicin-treated tumours compared with controls. *P < 0.05 relative to controls.

Mentions: HCCs are commonly resistant to treatment because the malignant cells survive chemotherapy [19]. To explore the possible role of CSC cells in the development of chemoresistance, cultured HSC-SR, HSC-DF, HepG2 and N-LSC cells were treated with sorafenib and doxorubicin and the IC50 was analysed by MTS assay. Doxorubicin has been considered to be the most effective single agent and the multi-kinase inhibitor sorafenib has recently been approved for the therapy of advanced HCC [22]. Sorafenib acts by inhibition of the RAF/MEK/ERK pathway and down-regulation of MCL-1, leading to a disruption of survival signals in HCC cells [22, 23]. As shown in Figure 4A–D, IC50s of HSCs in two chemotherapeutic reagents tested are significantly higher than HepG2 HCC cells as well as normal liver stem cells, suggesting that HCC stem cells have greater chemoresistance in vitro. In addition, HSC xenograft tumours in nude mice are more resistant to doxorubicin treatment than the control HCC xenografts (Fig. 4E). Enhanced CD133 and let-7a expressions are observed in doxorubicin-treated HSC xenograft tumours when compare to controls (Fig. 4F and G), further implicated a functional link between CD133/let-7a and tumour stem cell phenotypes.


Functional analysis of microRNAs in human hepatocellular cancer stem cells.

Meng F, Glaser SS, Francis H, DeMorrow S, Han Y, Passarini JD, Stokes A, Cleary JP, Liu X, Venter J, Kumar P, Priester S, Hubble L, Staloch D, Sharma J, Liu CG, Alpini G - J. Cell. Mol. Med. (2012)

HCC stem cells are resistant to conventional chemotherapy. (A, C) After serum starvation of cultured cells overnight, sorafenib and doxorubicin were added at various concentrations (10−3–10−10 M) and cell viability was assessed after 72 hrs. IC50 graph of high-content image analysis of human hepatocytes and HCC stem cells treated by sorafenib and doxorubicin was illustrated. Quantitative data of MTS assay in the sorafenib treated cells were calculated for IC50 with XLfit software, which is marked in the middle of the box. (B, D) IC50 of sorafenib and doxorubicin in hepato-cytes and HCC stem cells is illustrated in bar graph. HCC stem cells under either self-renewal (HSC-SR) or differentiation (HSC-DF) are more resistance to sorafenib and doxorubicin than HepG2 cells and normal liver stem cells. IC50 results are expressed as the mean ± S.E. of eight different experiments. (E) Groups of five SCID Berge mice were selected 8–10 weeks old per treatment group. The human HSC-SRs (1000) as well as parental HCC cells (5 × 106) were injected subcutaneously and allowed to engraft for 10 days. At day 10 the tumour was visible, and then the doxorubicin treatment was started and the mice received the desired concentration of the drug intraper-oteneal three times per week for 14 days. At the end of the 14 days the tumour was dissociated into single cell suspension and assayed with Almar Blue to determine percent inhibition of the drug. (F, G) Differential expression of CD133 and let-7a in vivo following treatment with doxorubicin by real-time PCR is shown. Data represent percentage change in expression in doxo-cibicin-treated tumours compared with controls. *P < 0.05 relative to controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3116063&req=5

fig04: HCC stem cells are resistant to conventional chemotherapy. (A, C) After serum starvation of cultured cells overnight, sorafenib and doxorubicin were added at various concentrations (10−3–10−10 M) and cell viability was assessed after 72 hrs. IC50 graph of high-content image analysis of human hepatocytes and HCC stem cells treated by sorafenib and doxorubicin was illustrated. Quantitative data of MTS assay in the sorafenib treated cells were calculated for IC50 with XLfit software, which is marked in the middle of the box. (B, D) IC50 of sorafenib and doxorubicin in hepato-cytes and HCC stem cells is illustrated in bar graph. HCC stem cells under either self-renewal (HSC-SR) or differentiation (HSC-DF) are more resistance to sorafenib and doxorubicin than HepG2 cells and normal liver stem cells. IC50 results are expressed as the mean ± S.E. of eight different experiments. (E) Groups of five SCID Berge mice were selected 8–10 weeks old per treatment group. The human HSC-SRs (1000) as well as parental HCC cells (5 × 106) were injected subcutaneously and allowed to engraft for 10 days. At day 10 the tumour was visible, and then the doxorubicin treatment was started and the mice received the desired concentration of the drug intraper-oteneal three times per week for 14 days. At the end of the 14 days the tumour was dissociated into single cell suspension and assayed with Almar Blue to determine percent inhibition of the drug. (F, G) Differential expression of CD133 and let-7a in vivo following treatment with doxorubicin by real-time PCR is shown. Data represent percentage change in expression in doxo-cibicin-treated tumours compared with controls. *P < 0.05 relative to controls.
Mentions: HCCs are commonly resistant to treatment because the malignant cells survive chemotherapy [19]. To explore the possible role of CSC cells in the development of chemoresistance, cultured HSC-SR, HSC-DF, HepG2 and N-LSC cells were treated with sorafenib and doxorubicin and the IC50 was analysed by MTS assay. Doxorubicin has been considered to be the most effective single agent and the multi-kinase inhibitor sorafenib has recently been approved for the therapy of advanced HCC [22]. Sorafenib acts by inhibition of the RAF/MEK/ERK pathway and down-regulation of MCL-1, leading to a disruption of survival signals in HCC cells [22, 23]. As shown in Figure 4A–D, IC50s of HSCs in two chemotherapeutic reagents tested are significantly higher than HepG2 HCC cells as well as normal liver stem cells, suggesting that HCC stem cells have greater chemoresistance in vitro. In addition, HSC xenograft tumours in nude mice are more resistant to doxorubicin treatment than the control HCC xenografts (Fig. 4E). Enhanced CD133 and let-7a expressions are observed in doxorubicin-treated HSC xenograft tumours when compare to controls (Fig. 4F and G), further implicated a functional link between CD133/let-7a and tumour stem cell phenotypes.

Bottom Line: Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion.Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion.In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Scott & White Digestive Disease Research Center, Texas A&M Health Science Center College of Medicine and Scott & White Hospital, Temple, TX 76504, USA.

Show MeSH
Related in: MedlinePlus