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Prostasin may contribute to chemoresistance, repress cancer cells in ovarian cancer, and is involved in the signaling pathways of CASP/PAK2-p34/actin.

Yan BX, Ma JX, Zhang J, Guo Y, Mueller MD, Remick SC, Yu JJ - Cell Death Dis (2014)

Bottom Line: In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance.In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells.Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, School of Medicine, Department of Basic Pharmaceutical Sciences, School of Pharmacy, and Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506, USA [2] IcesnowYanyan Bioscience Association, Beijing 00094, China.

ABSTRACT
Ovarian cancer is the deadliest of gynecologic cancers, largely due to the development of drug resistance in chemotherapy. Prostasin may have an essential role in the oncogenesis. In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance. Our cell cultural model investigation demonstrates prostasin has important roles in the development of drug resistance and cancer cell survival. Forced overexpression of prostasin in ovarian cancer cells greatly induces cell death (resulting in 99% cell death in a drug-resistant cell line and 100% cell death in other tested cell lines). In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells. In vivo studies indicate that forced overexpression of prostasin in drug-resistant cells greatly inhibits the growth of tumors and may partially reverse drug resistance. Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways. Thus, we introduce prostain as a potential target for treating/repressing some ovarian tumors and have begun to identify their relevant molecular targets in specific signaling pathways.

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Related in: MedlinePlus

Prostasin regulates CASPs-PAK2-p34 axis and thereafter downstream signaling. (a) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun in O432-RP-pro-O (O432-RP cells transfected with prostasin cDNA which express higher levels of prostasin) control O432-RP-C cells (O432-RP cells transfected with pCI-neo vector). Mlck, PAK2-p34, JNK, C-Jun expressions increase and β-actin decreases in O432-RP-pro-O cells when compared with control O432-RP-C. (b) Comparison of actin, mlck, and pak1 expression between O432-RP-pro-O and control O432-RP-C cells by real-time qPCR. mRNA levels of actin decreases and mlck and pak1 increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (c) mRNA levels of CASPs detection by real-time qPCR in O432-RP-pro-O cells and control O432-RP-C cells. Several CASP expression increase in O432-RP-pro-O cells compared with the controls. (d) Immunoblot of CASP3, 9, and 10 in O432-RP-pro-O and O432-RP-C cells. CASP3, 9, and 10 protein levels increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (e) CASP inhibitors block CASP activity and thus PAK2 cleavage. PAK2-p34 protein was examined by immunoblot after CASP inhibitors were added to the medium. PAK2-p34 was seen decreased or lost when CASPs were inhibited at several time points. (f) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun, CASP3, 9, and 10 in prostasin knockdown O432-pro-D cells and controls O432 (mock transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). Reverse expression patterns for these genes are revealed in O432-pro-D cells and controls compared with O432-RP-pro-O cells and controls
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fig4: Prostasin regulates CASPs-PAK2-p34 axis and thereafter downstream signaling. (a) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun in O432-RP-pro-O (O432-RP cells transfected with prostasin cDNA which express higher levels of prostasin) control O432-RP-C cells (O432-RP cells transfected with pCI-neo vector). Mlck, PAK2-p34, JNK, C-Jun expressions increase and β-actin decreases in O432-RP-pro-O cells when compared with control O432-RP-C. (b) Comparison of actin, mlck, and pak1 expression between O432-RP-pro-O and control O432-RP-C cells by real-time qPCR. mRNA levels of actin decreases and mlck and pak1 increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (c) mRNA levels of CASPs detection by real-time qPCR in O432-RP-pro-O cells and control O432-RP-C cells. Several CASP expression increase in O432-RP-pro-O cells compared with the controls. (d) Immunoblot of CASP3, 9, and 10 in O432-RP-pro-O and O432-RP-C cells. CASP3, 9, and 10 protein levels increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (e) CASP inhibitors block CASP activity and thus PAK2 cleavage. PAK2-p34 protein was examined by immunoblot after CASP inhibitors were added to the medium. PAK2-p34 was seen decreased or lost when CASPs were inhibited at several time points. (f) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun, CASP3, 9, and 10 in prostasin knockdown O432-pro-D cells and controls O432 (mock transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). Reverse expression patterns for these genes are revealed in O432-pro-D cells and controls compared with O432-RP-pro-O cells and controls

Mentions: β-Actin was found to be decreased in O432-RP cells compared with O432 cells in our study (we initially used β-actin as a loading control for western blot analysis; however, we found β-actin is not consistant when we used GAPDH as loading control for equal loading of samples (Figure 4a)). With the previous finding that β-actin expression changed in breast cancer drug resistance cells, this prompted us to hypothesize that β-actin and cytoskeletal genes may be involved in the prostasin-directed chemoresistance development as actin gene is believed to be a central player of cell shape and movement and a key component of cytoskeleton.33 We examined gene expression of cytoskeleton pathway using PCR array in these cells. The PCR array data showed that PAK and mlck increased, and β-actin decreased in O432-RP-pro-O cells, compared with control O432-RP-C cells (Figure 4b). Western blot analysis further demonstrated that protein levels of mlck and β-actin changed, which were consistent with mRNA levels (Figure 4a). However, we did not see significant difference for PAK proteins. Instead, we observed that PAK2-p34, a 34KD C-terminal fragment of PAK2, which is cleaved by CASPs,34, 35 is increased in O432-RP-pro-O cells. PAK2-p34 has been shown to regulate JNK expression during apoptosis,20 so we examined JNK and thereafter target c-jun expression. The western blot analysis showed that JNK and c-Jun both increased in O432-RP-pro-O cells. The data suggested that prostasin regulates PAK2-p34 and thereafter JNK and c-jun signaling in these cells. In addition, mlck has been shown to be a downstream target of PAK2/PAK2-p34, and upstream target of actin. Thus, PAK2-p34 seems to be an important mediator of prostasin in these cells and appears to regulate JNK/c-jun and mlck/act sub-pathways.


Prostasin may contribute to chemoresistance, repress cancer cells in ovarian cancer, and is involved in the signaling pathways of CASP/PAK2-p34/actin.

Yan BX, Ma JX, Zhang J, Guo Y, Mueller MD, Remick SC, Yu JJ - Cell Death Dis (2014)

Prostasin regulates CASPs-PAK2-p34 axis and thereafter downstream signaling. (a) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun in O432-RP-pro-O (O432-RP cells transfected with prostasin cDNA which express higher levels of prostasin) control O432-RP-C cells (O432-RP cells transfected with pCI-neo vector). Mlck, PAK2-p34, JNK, C-Jun expressions increase and β-actin decreases in O432-RP-pro-O cells when compared with control O432-RP-C. (b) Comparison of actin, mlck, and pak1 expression between O432-RP-pro-O and control O432-RP-C cells by real-time qPCR. mRNA levels of actin decreases and mlck and pak1 increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (c) mRNA levels of CASPs detection by real-time qPCR in O432-RP-pro-O cells and control O432-RP-C cells. Several CASP expression increase in O432-RP-pro-O cells compared with the controls. (d) Immunoblot of CASP3, 9, and 10 in O432-RP-pro-O and O432-RP-C cells. CASP3, 9, and 10 protein levels increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (e) CASP inhibitors block CASP activity and thus PAK2 cleavage. PAK2-p34 protein was examined by immunoblot after CASP inhibitors were added to the medium. PAK2-p34 was seen decreased or lost when CASPs were inhibited at several time points. (f) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun, CASP3, 9, and 10 in prostasin knockdown O432-pro-D cells and controls O432 (mock transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). Reverse expression patterns for these genes are revealed in O432-pro-D cells and controls compared with O432-RP-pro-O cells and controls
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043260&req=5

fig4: Prostasin regulates CASPs-PAK2-p34 axis and thereafter downstream signaling. (a) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun in O432-RP-pro-O (O432-RP cells transfected with prostasin cDNA which express higher levels of prostasin) control O432-RP-C cells (O432-RP cells transfected with pCI-neo vector). Mlck, PAK2-p34, JNK, C-Jun expressions increase and β-actin decreases in O432-RP-pro-O cells when compared with control O432-RP-C. (b) Comparison of actin, mlck, and pak1 expression between O432-RP-pro-O and control O432-RP-C cells by real-time qPCR. mRNA levels of actin decreases and mlck and pak1 increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (c) mRNA levels of CASPs detection by real-time qPCR in O432-RP-pro-O cells and control O432-RP-C cells. Several CASP expression increase in O432-RP-pro-O cells compared with the controls. (d) Immunoblot of CASP3, 9, and 10 in O432-RP-pro-O and O432-RP-C cells. CASP3, 9, and 10 protein levels increase in O432-RP-pro-O cells compared with control O432-RP-C cells. (e) CASP inhibitors block CASP activity and thus PAK2 cleavage. PAK2-p34 protein was examined by immunoblot after CASP inhibitors were added to the medium. PAK2-p34 was seen decreased or lost when CASPs were inhibited at several time points. (f) Immunoblot of mlck, β-actin, PAK2-p34, JNK, C-Jun, CASP3, 9, and 10 in prostasin knockdown O432-pro-D cells and controls O432 (mock transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). Reverse expression patterns for these genes are revealed in O432-pro-D cells and controls compared with O432-RP-pro-O cells and controls
Mentions: β-Actin was found to be decreased in O432-RP cells compared with O432 cells in our study (we initially used β-actin as a loading control for western blot analysis; however, we found β-actin is not consistant when we used GAPDH as loading control for equal loading of samples (Figure 4a)). With the previous finding that β-actin expression changed in breast cancer drug resistance cells, this prompted us to hypothesize that β-actin and cytoskeletal genes may be involved in the prostasin-directed chemoresistance development as actin gene is believed to be a central player of cell shape and movement and a key component of cytoskeleton.33 We examined gene expression of cytoskeleton pathway using PCR array in these cells. The PCR array data showed that PAK and mlck increased, and β-actin decreased in O432-RP-pro-O cells, compared with control O432-RP-C cells (Figure 4b). Western blot analysis further demonstrated that protein levels of mlck and β-actin changed, which were consistent with mRNA levels (Figure 4a). However, we did not see significant difference for PAK proteins. Instead, we observed that PAK2-p34, a 34KD C-terminal fragment of PAK2, which is cleaved by CASPs,34, 35 is increased in O432-RP-pro-O cells. PAK2-p34 has been shown to regulate JNK expression during apoptosis,20 so we examined JNK and thereafter target c-jun expression. The western blot analysis showed that JNK and c-Jun both increased in O432-RP-pro-O cells. The data suggested that prostasin regulates PAK2-p34 and thereafter JNK and c-jun signaling in these cells. In addition, mlck has been shown to be a downstream target of PAK2/PAK2-p34, and upstream target of actin. Thus, PAK2-p34 seems to be an important mediator of prostasin in these cells and appears to regulate JNK/c-jun and mlck/act sub-pathways.

Bottom Line: In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance.In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells.Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, School of Medicine, Department of Basic Pharmaceutical Sciences, School of Pharmacy, and Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506, USA [2] IcesnowYanyan Bioscience Association, Beijing 00094, China.

ABSTRACT
Ovarian cancer is the deadliest of gynecologic cancers, largely due to the development of drug resistance in chemotherapy. Prostasin may have an essential role in the oncogenesis. In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance. Our cell cultural model investigation demonstrates prostasin has important roles in the development of drug resistance and cancer cell survival. Forced overexpression of prostasin in ovarian cancer cells greatly induces cell death (resulting in 99% cell death in a drug-resistant cell line and 100% cell death in other tested cell lines). In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells. In vivo studies indicate that forced overexpression of prostasin in drug-resistant cells greatly inhibits the growth of tumors and may partially reverse drug resistance. Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways. Thus, we introduce prostain as a potential target for treating/repressing some ovarian tumors and have begun to identify their relevant molecular targets in specific signaling pathways.

Show MeSH
Related in: MedlinePlus