Limits...
SAG-UPS attenuates proapoptotic SARM and Noxa to confer survival advantage to early hepatocellular carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocellular carcinoma (HCC) is a deadly cancer because of its commonly late diagnosis and limited treatment options. SAG (sensitive to apoptosis gene)-dependent UPS (ubiquitin–proteasome system) is a key switch between immune-mediated apoptosis and overactivation-mediated protumorigenesis, prompting us to hypothesize that SAG-UPS modulates chronic inflammation-induced tumorigenesis. Here, we investigated the molecular mechanism by which SAG-UPS regulates death/survival of liver cancer cells. By retrospective studies, we found reciprocal expressions of anti-/proapoptotic factors: SAG/SARM and SAG/Noxa in human primary HCC tissues – the antiapoptotic SAG was significantly upregulated whereas the proapoptotic SARM and Noxa were markedly downregulated, suggesting their involvement in hepatocarcinogenesis. Upregulated SAG-UPS effectively manipulates the levels of high-molecular-weight ubiquitinated SARM and Noxa in carcinoma tissues compared with corresponding normal tissues. SAG-overexpressing HCC cell lines display reduced SARM and Noxa (but not Bcl-2, Bax and Bcl-xL), suggesting that SARM and Noxa are specific substrates of SAG-dependent ubiquitination. SARM overexpression activated caspase-3 and caspase-9, reducing cell viability. SAG knockdown significantly elevated apoptosis with increased cytosolic cytochrome c, confirming SAG-mediated antiapoptosis in HCC. SAG overexpression stimulated protumorigenic cytokines, IL-1β, IL-6 and TNF, but not antitumorigenic IL-12p40 and anti-inflammatory IL-10. This is consistent with higher proinflammatory cytokines (IL-1β, IL-6 and TNF) in hepatoma compared with healthy tissues. Altogether, early stage-upregulated SAG-UPS exacerbates hepatocarcinogenesis progression, through: (1) ubiquitination-mediated degradation of proapoptotic SARM and Noxa; and (2) production of protumorigenic cytokines that induce a protumorigenic microenvironment, conferring survival advantage to HCC cells. Thus, we propose SAG-UPS to be an early diagnostic marker for HCC, and a potential target for therapeutics development.

No MeSH data available.


Related in: MedlinePlus

SAG acts as an antiapoptotic and protumorigenic factor in human HCC. Six human HCC cell lines were transfected with SAG siRNA (or control siRNA) for 24 h, followed by (a) apoptosis analysis and (b) cytochrome c release assay. Apoptotic cells were determined by double staining with Annexin V and 7AAD. FACS analysis showed a significant increase in early apoptotic cells (Annexin V+/7AAD−) in SAG-knockdown cells. Cytochrome c release indicates the level of intrinsic apoptosis that is increased in all SAG-knockdown HCC cells. (c) Six HCC cells were transfected with SAG-HA-pcDNA or Control pcDNA for 24 h. The levels of protumorigenic cytokines (IL-1β, TNF and IL-6), antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10) in the culture supernatants were then examined using ELISA (n=3). (d) The mRNA levels of the cytokines were measured by real-time PCR in primary HCC tissues. C, carcinoma tissues; N, normal tissues. **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979479&req=5

fig5: SAG acts as an antiapoptotic and protumorigenic factor in human HCC. Six human HCC cell lines were transfected with SAG siRNA (or control siRNA) for 24 h, followed by (a) apoptosis analysis and (b) cytochrome c release assay. Apoptotic cells were determined by double staining with Annexin V and 7AAD. FACS analysis showed a significant increase in early apoptotic cells (Annexin V+/7AAD−) in SAG-knockdown cells. Cytochrome c release indicates the level of intrinsic apoptosis that is increased in all SAG-knockdown HCC cells. (c) Six HCC cells were transfected with SAG-HA-pcDNA or Control pcDNA for 24 h. The levels of protumorigenic cytokines (IL-1β, TNF and IL-6), antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10) in the culture supernatants were then examined using ELISA (n=3). (d) The mRNA levels of the cytokines were measured by real-time PCR in primary HCC tissues. C, carcinoma tissues; N, normal tissues. **P<0.01.

Mentions: FACS analysis (Supplementary Figure 6) showed that SAG knockdown significantly increased apoptosis in all six HCC cell lines (Figure 5a, P<0.01). At 24 h post siRNA treatment, early apoptosis was observed in 35-40% of Chang and Huh1 cells compared with 10–25% of Huh7, Hep3B, HepG2 and SNU449 cell lines. Control siRNA (or PBS)-treated cells showed minimal apoptosis, suggesting that the increased apoptosis of HCC cells is specifically induced by SAG silencing.


SAG-UPS attenuates proapoptotic SARM and Noxa to confer survival advantage to early hepatocellular carcinoma
SAG acts as an antiapoptotic and protumorigenic factor in human HCC. Six human HCC cell lines were transfected with SAG siRNA (or control siRNA) for 24 h, followed by (a) apoptosis analysis and (b) cytochrome c release assay. Apoptotic cells were determined by double staining with Annexin V and 7AAD. FACS analysis showed a significant increase in early apoptotic cells (Annexin V+/7AAD−) in SAG-knockdown cells. Cytochrome c release indicates the level of intrinsic apoptosis that is increased in all SAG-knockdown HCC cells. (c) Six HCC cells were transfected with SAG-HA-pcDNA or Control pcDNA for 24 h. The levels of protumorigenic cytokines (IL-1β, TNF and IL-6), antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10) in the culture supernatants were then examined using ELISA (n=3). (d) The mRNA levels of the cytokines were measured by real-time PCR in primary HCC tissues. C, carcinoma tissues; N, normal tissues. **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979479&req=5

fig5: SAG acts as an antiapoptotic and protumorigenic factor in human HCC. Six human HCC cell lines were transfected with SAG siRNA (or control siRNA) for 24 h, followed by (a) apoptosis analysis and (b) cytochrome c release assay. Apoptotic cells were determined by double staining with Annexin V and 7AAD. FACS analysis showed a significant increase in early apoptotic cells (Annexin V+/7AAD−) in SAG-knockdown cells. Cytochrome c release indicates the level of intrinsic apoptosis that is increased in all SAG-knockdown HCC cells. (c) Six HCC cells were transfected with SAG-HA-pcDNA or Control pcDNA for 24 h. The levels of protumorigenic cytokines (IL-1β, TNF and IL-6), antitumorigenic cytokine (IL-12p40) and anti-inflammatory cytokine (IL-10) in the culture supernatants were then examined using ELISA (n=3). (d) The mRNA levels of the cytokines were measured by real-time PCR in primary HCC tissues. C, carcinoma tissues; N, normal tissues. **P<0.01.
Mentions: FACS analysis (Supplementary Figure 6) showed that SAG knockdown significantly increased apoptosis in all six HCC cell lines (Figure 5a, P<0.01). At 24 h post siRNA treatment, early apoptosis was observed in 35-40% of Chang and Huh1 cells compared with 10–25% of Huh7, Hep3B, HepG2 and SNU449 cell lines. Control siRNA (or PBS)-treated cells showed minimal apoptosis, suggesting that the increased apoptosis of HCC cells is specifically induced by SAG silencing.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocellular carcinoma (HCC) is a deadly cancer because of its commonly late diagnosis and limited treatment options. SAG (sensitive to apoptosis gene)-dependent UPS (ubiquitin&ndash;proteasome system) is a key switch between immune-mediated apoptosis and overactivation-mediated protumorigenesis, prompting us to hypothesize that SAG-UPS modulates chronic inflammation-induced tumorigenesis. Here, we investigated the molecular mechanism by which SAG-UPS regulates death/survival of liver cancer cells. By retrospective studies, we found reciprocal expressions of anti-/proapoptotic factors: SAG/SARM and SAG/Noxa in human primary HCC tissues &ndash; the antiapoptotic SAG was significantly upregulated whereas the proapoptotic SARM and Noxa were markedly downregulated, suggesting their involvement in hepatocarcinogenesis. Upregulated SAG-UPS effectively manipulates the levels of high-molecular-weight ubiquitinated SARM and Noxa in carcinoma tissues compared with corresponding normal tissues. SAG-overexpressing HCC cell lines display reduced SARM and Noxa (but not Bcl-2, Bax and Bcl-xL), suggesting that SARM and Noxa are specific substrates of SAG-dependent ubiquitination. SARM overexpression activated caspase-3 and caspase-9, reducing cell viability. SAG knockdown significantly elevated apoptosis with increased cytosolic cytochrome c, confirming SAG-mediated antiapoptosis in HCC. SAG overexpression stimulated protumorigenic cytokines, IL-1&beta;, IL-6 and TNF, but not antitumorigenic IL-12p40 and anti-inflammatory IL-10. This is consistent with higher proinflammatory cytokines (IL-1&beta;, IL-6 and TNF) in hepatoma compared with healthy tissues. Altogether, early stage-upregulated SAG-UPS exacerbates hepatocarcinogenesis progression, through: (1) ubiquitination-mediated degradation of proapoptotic SARM and Noxa; and (2) production of protumorigenic cytokines that induce a protumorigenic microenvironment, conferring survival advantage to HCC cells. Thus, we propose SAG-UPS to be an early diagnostic marker for HCC, and a potential target for therapeutics development.

No MeSH data available.


Related in: MedlinePlus