Limits...
Genomic analysis of xCT-mediated regulatory network: Identification of novel targets against AIDS-associated lymphoma.

Dai L, Cao Y, Chen Y, Kaleeba JA, Zabaleta J, Qin Z - Oncotarget (2015)

Bottom Line: We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors.More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model.Together, our data indicate that targeting these novel xCT-regulated downstream genes may represent a promising new therapeutic strategy against PEL and/or other AIDS-related lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Translational Medicine and Key Laboratory of Arrhythmias of The Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), a rapidly progressing malignancy mostly arising in HIV-infected patients. Even under conventional chemotherapy, PEL continues to portend nearly 100% mortality within several months, which urgently requires novel therapeutic strategies. We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors. More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model. In the current study, we use Illumina microarray to explore the profile of genes altered by SASP treatment within 3 KSHV(+) PEL cell-lines, and discover that many genes involved in oxidative stress/antioxidant defense system, apoptosis/anti-apoptosis/cell death, and cellular response to unfolded proteins/topologically incorrect proteins are potentially regulated by xCT. We further validate 2 downstream candidates, OSGIN1 (oxidative stress-induced growth inhibitor 1) and XRCC5 (X-ray repair cross-complementing protein 5), and evaluate their functional relationship with PEL cell survival/proliferation and chemoresistance, respectively. Together, our data indicate that targeting these novel xCT-regulated downstream genes may represent a promising new therapeutic strategy against PEL and/or other AIDS-related lymphoma.

No MeSH data available.


Related in: MedlinePlus

xCT inhibition induces KSHV-infected PEL cell apoptosis potentially through upregulation of OSGIN1(A) BCP-1 and BCBL-1 were transfected with either negative control siRNA (n-siRNA) or OSGIN1-siRNA for 48 h, then incubated with 0.5mM of SASP for additional 24 h and cell apoptosis was assessed using Annexin V-PI staining and flow cytometry analysis. (B) Protein expression in BCBL-1 was measured by immuoblots. (C-D) The levels of intracellular GSH and ROS were quantified as described in Methods. (E) The activities of NADPH oxidases in BCBL-1 were measured as described in Methods. Error bars represent the S.E.M. for 3 independent experiments. **/## = p < 0.01 (vs SASP+n-siRNA group).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494968&req=5

Figure 5: xCT inhibition induces KSHV-infected PEL cell apoptosis potentially through upregulation of OSGIN1(A) BCP-1 and BCBL-1 were transfected with either negative control siRNA (n-siRNA) or OSGIN1-siRNA for 48 h, then incubated with 0.5mM of SASP for additional 24 h and cell apoptosis was assessed using Annexin V-PI staining and flow cytometry analysis. (B) Protein expression in BCBL-1 was measured by immuoblots. (C-D) The levels of intracellular GSH and ROS were quantified as described in Methods. (E) The activities of NADPH oxidases in BCBL-1 were measured as described in Methods. Error bars represent the S.E.M. for 3 independent experiments. **/## = p < 0.01 (vs SASP+n-siRNA group).

Mentions: We next selected OSGIN1 (Oxidative stress-induced growth inhibitor 1), one of the highly upregulated genes in SASP-treated KSHV+ PEL cells from microarray data, to determine its role in SASP-induced cell apoptosis. The OSGIN1 gene (also named as OKL38) was first identified in breast epithelial cells as increasingly expressed during pregnancy and lactation [24]. Low-level expression of this gene has been reported in breast cancer cell lines, while its overexpression in MCF-7 breast cancer cells leads to a reduction in proliferation as well as tumor formation in nude mice [24]. As a tumor suppressor, downregulation of OSGIN1 was also found to be closely associated with progression of other malignancies such as hepatocellular carcinoma and kidney cancer [25-27]. Here, we found that silencing of OSGIN1 by RNAi significantly reduced cell apoptosis induced by SASP (0.5 mM) in BCP-1 and BCBL-1 cells (Figures 5A and S2). Western blot analysis also indicated that silencing of OSGIN1 by RNAi in SASP-treated BCP-1 and BCBL-1 greatly reduced cleaved Caspase 3 and 9, while partially rescuing the phosphorylation of Akt, downstream P70S6, S6 and the expression of X-linked inhibitor of apoptosis protein (XIAP) [28], a physiologic substrate of Akt that is stabilized to inhibit programmed cell death (Figure 5B). We have previously shown that SASP-induced PEL apoptosis may also be orchestrated via reduction of intracellular GSH synthesis and increased ROS production [5]. Here we found that silencing of OSGIN1 significantly restored intracellular GSH synthesis and reduced ROS production from SASP-treated cells (Figure 5C-5D). Biochemical assays further confirmed that silencing of OSGIN1 caused a reduction in the activity of NADPH oxidase (Figure 5E), the major source of ROS production [29, 30]. We also found that silencing of xCT by RNAi upregulated OSGIN1 transcripts in BCBL-1 cells, indicating that this gene is indeed a downstream target of xCT (Figure S3A). Taken together, these data demonstrate the central role of OSGIN1 in SASP-induced PEL apoptosis, which involves modulation of a variety of host physiologic factors.


Genomic analysis of xCT-mediated regulatory network: Identification of novel targets against AIDS-associated lymphoma.

Dai L, Cao Y, Chen Y, Kaleeba JA, Zabaleta J, Qin Z - Oncotarget (2015)

xCT inhibition induces KSHV-infected PEL cell apoptosis potentially through upregulation of OSGIN1(A) BCP-1 and BCBL-1 were transfected with either negative control siRNA (n-siRNA) or OSGIN1-siRNA for 48 h, then incubated with 0.5mM of SASP for additional 24 h and cell apoptosis was assessed using Annexin V-PI staining and flow cytometry analysis. (B) Protein expression in BCBL-1 was measured by immuoblots. (C-D) The levels of intracellular GSH and ROS were quantified as described in Methods. (E) The activities of NADPH oxidases in BCBL-1 were measured as described in Methods. Error bars represent the S.E.M. for 3 independent experiments. **/## = p < 0.01 (vs SASP+n-siRNA group).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494968&req=5

Figure 5: xCT inhibition induces KSHV-infected PEL cell apoptosis potentially through upregulation of OSGIN1(A) BCP-1 and BCBL-1 were transfected with either negative control siRNA (n-siRNA) or OSGIN1-siRNA for 48 h, then incubated with 0.5mM of SASP for additional 24 h and cell apoptosis was assessed using Annexin V-PI staining and flow cytometry analysis. (B) Protein expression in BCBL-1 was measured by immuoblots. (C-D) The levels of intracellular GSH and ROS were quantified as described in Methods. (E) The activities of NADPH oxidases in BCBL-1 were measured as described in Methods. Error bars represent the S.E.M. for 3 independent experiments. **/## = p < 0.01 (vs SASP+n-siRNA group).
Mentions: We next selected OSGIN1 (Oxidative stress-induced growth inhibitor 1), one of the highly upregulated genes in SASP-treated KSHV+ PEL cells from microarray data, to determine its role in SASP-induced cell apoptosis. The OSGIN1 gene (also named as OKL38) was first identified in breast epithelial cells as increasingly expressed during pregnancy and lactation [24]. Low-level expression of this gene has been reported in breast cancer cell lines, while its overexpression in MCF-7 breast cancer cells leads to a reduction in proliferation as well as tumor formation in nude mice [24]. As a tumor suppressor, downregulation of OSGIN1 was also found to be closely associated with progression of other malignancies such as hepatocellular carcinoma and kidney cancer [25-27]. Here, we found that silencing of OSGIN1 by RNAi significantly reduced cell apoptosis induced by SASP (0.5 mM) in BCP-1 and BCBL-1 cells (Figures 5A and S2). Western blot analysis also indicated that silencing of OSGIN1 by RNAi in SASP-treated BCP-1 and BCBL-1 greatly reduced cleaved Caspase 3 and 9, while partially rescuing the phosphorylation of Akt, downstream P70S6, S6 and the expression of X-linked inhibitor of apoptosis protein (XIAP) [28], a physiologic substrate of Akt that is stabilized to inhibit programmed cell death (Figure 5B). We have previously shown that SASP-induced PEL apoptosis may also be orchestrated via reduction of intracellular GSH synthesis and increased ROS production [5]. Here we found that silencing of OSGIN1 significantly restored intracellular GSH synthesis and reduced ROS production from SASP-treated cells (Figure 5C-5D). Biochemical assays further confirmed that silencing of OSGIN1 caused a reduction in the activity of NADPH oxidase (Figure 5E), the major source of ROS production [29, 30]. We also found that silencing of xCT by RNAi upregulated OSGIN1 transcripts in BCBL-1 cells, indicating that this gene is indeed a downstream target of xCT (Figure S3A). Taken together, these data demonstrate the central role of OSGIN1 in SASP-induced PEL apoptosis, which involves modulation of a variety of host physiologic factors.

Bottom Line: We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors.More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model.Together, our data indicate that targeting these novel xCT-regulated downstream genes may represent a promising new therapeutic strategy against PEL and/or other AIDS-related lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Translational Medicine and Key Laboratory of Arrhythmias of The Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China.

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), a rapidly progressing malignancy mostly arising in HIV-infected patients. Even under conventional chemotherapy, PEL continues to portend nearly 100% mortality within several months, which urgently requires novel therapeutic strategies. We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors. More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model. In the current study, we use Illumina microarray to explore the profile of genes altered by SASP treatment within 3 KSHV(+) PEL cell-lines, and discover that many genes involved in oxidative stress/antioxidant defense system, apoptosis/anti-apoptosis/cell death, and cellular response to unfolded proteins/topologically incorrect proteins are potentially regulated by xCT. We further validate 2 downstream candidates, OSGIN1 (oxidative stress-induced growth inhibitor 1) and XRCC5 (X-ray repair cross-complementing protein 5), and evaluate their functional relationship with PEL cell survival/proliferation and chemoresistance, respectively. Together, our data indicate that targeting these novel xCT-regulated downstream genes may represent a promising new therapeutic strategy against PEL and/or other AIDS-related lymphoma.

No MeSH data available.


Related in: MedlinePlus