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JNK is required for maintaining the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Liu Y, Zhang X, Wang J, Yang J, Tan WF - Acta Pharmacol. Sin. (2015)

Bottom Line: Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells.Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity.Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.

ABSTRACT

Aim: Many studies reveal an association between the acquired chemoresistant phenotype of cancer cells and tumor-initiating cell-like properties. The aim of this study was to determine the impact of c-Jun N-terminal kinase (JNK) on the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Methods: Two well-established human acquired chemoresistant cancer cell lines K562/A02 and KB/VCR, as well as their respective parental counterparts K562 and KB were tested. The expression of relevant mRNAs and proteins was detected using qRT-PCR and Western blotting, respectively. Sphere formation and self-renewal assays were used to study the tumor-initiating cell-like properties. Soft agar and colony formation assays were used to investigate tumorigenic ability.

Results: We observed that suppressing JNK activity by its specific small molecule inhibitor SP600125 or by limiting JNK1/2 expression with JNK1/2 shRNA lentiviruses inhibited the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog in KB/VCR cells and K562/A02 cells as well as sphere formation and self-renewal abilities of K562/A02 cells. Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells. Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity.

Conclusion: JNK maintains the tumor-initiating cell-like properties of acquired chemoresistant K562/A02 and KB/VCR cells potentially through activating the Hedgehog pathway. Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.

No MeSH data available.


Related in: MedlinePlus

Inhibition of JNK activity suppresses stem cell markers of acquired chemoresistant cancer cells. (A–C) After treatment with or without SP600125 (10 μmol/L) for 24 h, KB and KB/VCR cells were collected for qRT-PCR and Western blot analyses. Treatment with SP600125 reduced the expression of Oct4 (A), Sox2 (B), and Nanog (C) at the mRNA level. (D) Western blot analysis of p-c-Jun, Oct4, Sox2, and Nanog expression in KB and KB/VCR cells treated with or without SP600125. (E) JNK, p-c-Jun, GFP, Oct4, Sox2, and Nanog immunoblots in K562/A02 cells that had been infected with shRNA control or JNK1/2 shRNA lentiviruses. Mean±SD. n=3.
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fig1: Inhibition of JNK activity suppresses stem cell markers of acquired chemoresistant cancer cells. (A–C) After treatment with or without SP600125 (10 μmol/L) for 24 h, KB and KB/VCR cells were collected for qRT-PCR and Western blot analyses. Treatment with SP600125 reduced the expression of Oct4 (A), Sox2 (B), and Nanog (C) at the mRNA level. (D) Western blot analysis of p-c-Jun, Oct4, Sox2, and Nanog expression in KB and KB/VCR cells treated with or without SP600125. (E) JNK, p-c-Jun, GFP, Oct4, Sox2, and Nanog immunoblots in K562/A02 cells that had been infected with shRNA control or JNK1/2 shRNA lentiviruses. Mean±SD. n=3.

Mentions: To determine whether JNK interference may disrupt the tumor-initiating cell-like properties of acquired chemoresistant cancer cells, we utilized SP600125, a specific small molecule JNK inhibitor12 to inhibit JNK activity. We first examined the effect of SP600125 on the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog, which are frequently expressed in poorly differentiated tumors and are recognized as markers of tumor-initiating cells13,14. qRT-PCR assays revealed that after SP600125 treatment, Oct4, Sox2, and Nanog expression was clearly suppressed in KB/VCR cells, while SP600125 had no effect on the expression of these stem markers in KB cells (Figure 1A–1C). Concomitantly, SP600125 also inhibited Oct4, Sox2, and Nanog protein expression in KB/VCR cells as revealed by Western blot analyses (Figure 1D). Similarly, we also limited JNK1/2 expression in K562/A02 by JNK1/2 shRNA lentiviruses to inhibit JNK activity. GFP blots were used as readouts of lentivirus infection efficiency, and p-c-Jun expression served as reduction in JNK activity. we observed that limiting JNK1/2 expression by shRNA in K562/A02 cells reduced Oct4, Sox2, and Nanog protein expression (Figure 1E).


JNK is required for maintaining the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Liu Y, Zhang X, Wang J, Yang J, Tan WF - Acta Pharmacol. Sin. (2015)

Inhibition of JNK activity suppresses stem cell markers of acquired chemoresistant cancer cells. (A–C) After treatment with or without SP600125 (10 μmol/L) for 24 h, KB and KB/VCR cells were collected for qRT-PCR and Western blot analyses. Treatment with SP600125 reduced the expression of Oct4 (A), Sox2 (B), and Nanog (C) at the mRNA level. (D) Western blot analysis of p-c-Jun, Oct4, Sox2, and Nanog expression in KB and KB/VCR cells treated with or without SP600125. (E) JNK, p-c-Jun, GFP, Oct4, Sox2, and Nanog immunoblots in K562/A02 cells that had been infected with shRNA control or JNK1/2 shRNA lentiviruses. Mean±SD. n=3.
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getmorefigures.php?uid=PMC4561974&req=5

fig1: Inhibition of JNK activity suppresses stem cell markers of acquired chemoresistant cancer cells. (A–C) After treatment with or without SP600125 (10 μmol/L) for 24 h, KB and KB/VCR cells were collected for qRT-PCR and Western blot analyses. Treatment with SP600125 reduced the expression of Oct4 (A), Sox2 (B), and Nanog (C) at the mRNA level. (D) Western blot analysis of p-c-Jun, Oct4, Sox2, and Nanog expression in KB and KB/VCR cells treated with or without SP600125. (E) JNK, p-c-Jun, GFP, Oct4, Sox2, and Nanog immunoblots in K562/A02 cells that had been infected with shRNA control or JNK1/2 shRNA lentiviruses. Mean±SD. n=3.
Mentions: To determine whether JNK interference may disrupt the tumor-initiating cell-like properties of acquired chemoresistant cancer cells, we utilized SP600125, a specific small molecule JNK inhibitor12 to inhibit JNK activity. We first examined the effect of SP600125 on the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog, which are frequently expressed in poorly differentiated tumors and are recognized as markers of tumor-initiating cells13,14. qRT-PCR assays revealed that after SP600125 treatment, Oct4, Sox2, and Nanog expression was clearly suppressed in KB/VCR cells, while SP600125 had no effect on the expression of these stem markers in KB cells (Figure 1A–1C). Concomitantly, SP600125 also inhibited Oct4, Sox2, and Nanog protein expression in KB/VCR cells as revealed by Western blot analyses (Figure 1D). Similarly, we also limited JNK1/2 expression in K562/A02 by JNK1/2 shRNA lentiviruses to inhibit JNK activity. GFP blots were used as readouts of lentivirus infection efficiency, and p-c-Jun expression served as reduction in JNK activity. we observed that limiting JNK1/2 expression by shRNA in K562/A02 cells reduced Oct4, Sox2, and Nanog protein expression (Figure 1E).

Bottom Line: Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells.Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity.Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.

ABSTRACT

Aim: Many studies reveal an association between the acquired chemoresistant phenotype of cancer cells and tumor-initiating cell-like properties. The aim of this study was to determine the impact of c-Jun N-terminal kinase (JNK) on the tumor-initiating cell-like properties of acquired chemoresistant human cancer cells.

Methods: Two well-established human acquired chemoresistant cancer cell lines K562/A02 and KB/VCR, as well as their respective parental counterparts K562 and KB were tested. The expression of relevant mRNAs and proteins was detected using qRT-PCR and Western blotting, respectively. Sphere formation and self-renewal assays were used to study the tumor-initiating cell-like properties. Soft agar and colony formation assays were used to investigate tumorigenic ability.

Results: We observed that suppressing JNK activity by its specific small molecule inhibitor SP600125 or by limiting JNK1/2 expression with JNK1/2 shRNA lentiviruses inhibited the expression of pluripotent stem cell markers such as Oct4, Sox2, and Nanog in KB/VCR cells and K562/A02 cells as well as sphere formation and self-renewal abilities of K562/A02 cells. Additionally, inhibition of JNK activity significantly inhibited the in vitro and in vivo tumor-initiating abilities of KB/VCR cells. Furthermore, our data suggest that blocking JNK activity abundantly inhibited the Hedgehog (Hh) pathway activity, as reflected by reduction of Hedgehog (Hh) pathway target genes Gli1 and ptch1 at the mRNA level as well as Gli-luciferase activity.

Conclusion: JNK maintains the tumor-initiating cell-like properties of acquired chemoresistant K562/A02 and KB/VCR cells potentially through activating the Hedgehog pathway. Thus, disruption of tumor-initiating cell-like properties by targeting JNK may be a new approach to combating acquired chemoresistance.

No MeSH data available.


Related in: MedlinePlus