Xiaowu Pang

Enforced Expression of miR-101 Inhibits Prostate Cancer Cell Growth by Modulating the COX-2 Pathway In Vivo

It is commonly agreed that there is an association of chronic inflammation with tumorigenesis. COX-2, a key regulator of inflammation-producing prostaglandins, promotes cell proliferation and growth; thus, overexpression of COX-2 is often found in tumor tissues. Therefore, a better understanding of the regulatory mechanism(s) of COX-2 could lead to novel targeted cancer therapies. In this study, we investigated the mechanism of microRNA-101 (miR-101)-regulated COX-2 expression and the therapeutic potential of exogenous miR-101 for COX-2–associated cancer. A stably expressing exogenous miR-101 prostate cancer cell line (BPH1CmiR101) was generated by using lentiviral transduction as a tool for in vitro and in vivo studies. We found that miR-101 inhibited COX-2 posttranscriptional expression by directly binding to the 3′-untranslated region (3′-UTR) of COX-2 mRNA. The regulatory function of miR-101 was also confirmed by using antisense DNA. As a result, exogenous miR-101 is able to effectively suppress the growth of cultured prostate cancer cells and prostate tumor xenografts. The average tumor weight was significantly lower in the BPH1CmiR101 group (0.22 g) than the BPH1Cvec group (0.46 g). Expression levels of the cell growth regulators, such as cyclin proteins, PCNA (proliferating cell nuclear antigen), EGFR (epidermal growth factor receptor), were also studied. In conclusion, COX-2 is a direct target in miR-101 regulation of posttranscription. Exogenous miR-101 suppresses the proliferation and growth of prostate cancer cells in vitro and in vivo. These data suggest that exogenous miR-101 may provide a new cancer therapy by directly inhibiting COX-2 expression. Cancer Prev Res; 4(7); 1073–83. ©2011 AACR.

Vitamin E Succinate Induces Ceramide-Mediated Apoptosis in Head and Neck Squamous Cell Carcinoma In vitro and In vivo

Purpose: Vitamin E succinate (α-TOS) inhibits the growth of cancer cells without unacceptable side effects. Therefore, the mechanisms associated with the anticancer action of α-TOS, including ceramide-mediated apoptosis, were investigated using head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Experimental Design: Five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) were treated with α-TOS, and its effects on cell proliferation, cell cycle progression, ceramide-mediated apoptosis, and ceramide metabolism were evaluated. The anticancer effect of α-TOS was also examined on JHU-022 solid tumor xenograft growth in immunodeficient mice. Results: α-TOS inhibited the growth of all the HNSCC cell lines in vitro in a dose- and time-dependent manner. Thus, JHU-013 and JHU-022 cell lines were more sensitive to α-TOS than the other cell lines. Cellular levels of ceramide, sphingomyelinase activity, caspase-3, and p53 were elevated with increasing time of exposure to α-TOS. The degradation of poly(ADP-ribose) polymerase protein in JHU-022 cells treated with α-TOS provided evidence for apoptosis. The amounts of nuclear factor κB, Bcl-2, and Bcl-XL proteins were reduced in the cells treated with α-TOS for 6 hours. The levels of caspase-9, murine double minute-2, and IκB-α proteins were unchanged after α-TOS treatment. I.p. administration of α-TOS slowed tumor growth in immunodeficient mice. Conclusions: α-TOS showed promising anticancer effects to inhibit HNSCC growth and viability in vivo and in vitro. The induction of enzymes involved in ceramide metabolism by α-TOS suggests that ceramide-mediated apoptosis may expand therapeutic strategies in the treatment of carcinoma.

Salvianolic acid B inhibits growth of head and neck squamous cell carcinoma in vitro and in vivo via cyclooxygenase-2 and apoptotic pathways

Overexpression of cyclooxygenase‐2 (COX‐2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti‐inflammatory drug, which inhibits COX‐2 but not COX‐1. This selective COX‐2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long‐term chemoprevention and therapy. Salvianolic acid B (Sal‐B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal‐B inhibits HNSCC growth. Sal‐B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal‐B was assessed in HNSCC and other cell lines by estimating COX‐2 expression, cell viability and caspase‐dependent apoptosis. Sal‐B inhibited growth of HNSCC JHU‐022 and JHU‐013 cells with IC50 of 18 and 50 μM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal‐B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX‐2 inhibitors. Tumor volumes in Sal‐B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal‐B inhibited COX‐2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal‐B also caused dose‐dependent inhibition of prostaglandin E2 synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal‐B shows promise as a COX‐2 targeted anticancer agent for HNSCC prevention and treatment.

Combination Effects of Salvianolic Acid B with Low Dose Celecoxib on Inhibition of Head and Neck Squamous Cell Carcinoma Growth in vitro and in vivo

Head and neck squamous cell carcinoma (HNSCC) development is closely associated with inflammation. Cyclooxygenase-2 (COX-2) is an important mediator of inflammation. Therefore, celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib, but it seems likely that this may be diminished by lowering its dose. We found that salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, can effectively suppress COX-2 expression and induce apoptosis in a variety of cancer cell lines. In this study, we report that combination of Sal-B with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. The combination effects were assessed in four HNSCC cell lines (JHU-06, JHU-011, JHU-013, and JHU-022) by evaluating cell viability, proliferation, and tumor xenograft growth. Cell viability and proliferation were significantly inhibited by both the combined and single-agent treatments. However, the combination treatment significantly enhanced anticancer efficacy in JHU-013 and JHU-022 cell lines compared with the single treatment regimens. A half-dose of daily Sal-B (40 mg/kg/d) and celecoxib (2.5 mg/kg/d) significantly inhibited JHU-013 xenograft growth relative to mice treated with a full dose of Sal-B or celecoxib alone. The combination was associated with profound inhibition of COX-2 and enhanced induction of apoptosis. Taken together, these results strongly suggest that combination of Sal-B, a multifunctional anticancer agent, with low-dose celecoxib holds potential as a new preventive strategy in targeting inflammatory-associated tumor development. Cancer Prev Res; 3(6); 787–96. ©2010 AACR.

Improvement of Prostate Cancer Detection by Integrating the PSA Test With miRNA Expression Profiling

Prostate-specific antigen (PSA) test is limited in prostate cancer diagnosis due to its inaccuracy. A new approach which integrates the PSA test with miRNA profiling was investigated to improve prostate cancer diagnosis. Six prostate cancer-related miRNAs (miR-16, -21, -34c, -101, -125b, -141) were tested in five cultured prostate cell lines and 20 human prostate specimens. We found that the miRNA expression profiles were significantly different between nontumorigenic and tumorigenic cell lines and specimens. Positive predictive value analysis of prostate cancer was increased from 40% to 87.5% by integrating patient PSA blood levels with miR-21 and miR-141 profiles.

Functions of MiRNA-128 on the regulation of head and neck squamous cell carcinoma growth and apoptosis.

Background Incidence of head and neck squamous cell carcinoma (HNSCC) has continuously increased in past years while its survival rate has not been significantly improved. There is a critical need to better understand the genetic regulation of HNSCC tumorigenesis and progression. In this study, we comprehensively analyzed the function of miRNA-128 (miR-128) in the regulation of HNSCC growth and its putative targets in vitro and in vivo systems. Methods The function and targets of miR-128 were investigated in human HNSCC cell lines (JHU-13 and JHU-22), which were stably transfected with the miR-128 gene using a lentiviral delivery system. The expression levels of miR-128 and its targeted proteins were analyzed with qRT-PCR, Western blotting and flow cytometry. The binding capacity of miRNA-128 to its putative targets was determined using a luciferase report assay. MTT, colony formation, and a tumor xenograft model further evaluated the effects of miR-128 on HNSCC growth. Results We generated two miR-128 stably transfected human HNSCC cell lines (JHU-13miR-128 and JHU-22miR-128). Enforced expression of miR-128 was detected in both cultured JHU-13miR-128 and JHU-22miR-128 cell lines, approximately seventeen to twenty folds higher than in vector control cell lines. miRNA-128 was able to bind with the 3′-untranslated regions of BMI-1, BAG-2, BAX, H3f3b, and Paip2 mRNAs, resulting in significant reduction of the targeted protein levels. We found that upregulated miR-128 expression significantly inhibited both JHU-13miR-128 and JHU-22miR-128 cell viability approximately 20 to 40%, and the JHU-22miR-128 tumor xenograft growth compared to the vector control groups. Conclusions miR-128 acted as a tumor suppressor inhibiting the HNSCC growth by directly mediating the expression of putative targets. Our results provide a better understanding of miRNA-128 function and its potential targets, which may be valuable for developing novel diagnostic markers and targeted therapy.

MiR-124 acts as a tumor suppressor by inhibiting the expression of sphingosine kinase 1 and its downstream signaling in head and neck squamous cell carcinoma

By analyzing the expression profile of microRNAs in head and neck squamous cell carcinomas (HNSCC), we found that the expression level of miR-124 was 4.59-fold lower in tumors than in normal tissues. To understand its functions, we generated a miR-124-expressing subline (JHU-22miR124) and a mock vector-transfected subline (JHU-22vec) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22miR124 cells led to reduced cell proliferation, delayed colony formation, and decreased tumor growth, indicating a tumor-suppressive effect of miR-124. Subsequent target search revealed that the 3′-UTR of SphK1 mRNA carries a complementary site for the seed region of miR-124. SphK1 was also detected to be overexpressed in HNSCC cell lines, but down-expressed in JHU-22miR124 cells and tumor xenografts. These results suggest that SphK1 is a target of miR-124. To confirm this finding, we constructed a 3′-UTR-Luc-SphK1 vector and a binding site-mutated luciferase reporter vector. Co-transfection of 3′-UTR-Luc-SphK1 with miR-124 expression vector exhibited a 9-fold decrease in luciferase activity compared with mutated vector, suggesting that miR-124 inhibits SphK1 activity directly. Further studies on downstream signaling demonstrated accumulation of ceramide, increased expression of the pro-apoptotic Bax, BAD and PARP, decreased expression of the anti-apoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22miR124 compared with JHU-22vec cells and tumor xenografts. We conclude that miR-124 acts as a tumor suppressor in HNSCC by directly inhibiting SphK1 activity and its downstream signals.

Abstract 3170: Comprehensive analysis of the microRNA-128 function in head and neck squamous cell carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MicroRNAs are 22∼nucleotide-long endogenously expressed, highly conserved noncoding RNAs with important regulatory functions in proliferation, apoptosis, and metastasis. miR-128 a brain-enriched, which is transcribes from two gene copies (miR-128-1 and miR-128-2) on chromosome 2 and chromosome 3 which produce identical mature forms. miR-128 is most commonly in gliomas that have shown to function as a tumor suppressor which inhibits cell proliferation. Proteomic alterations of prostate cancer show miR-128 to down regulate cell invasiveness. Here we evaluated the role of miRNA-128 in HNSCC, to identify its putative targets and elucidate the possible mechanisms underlining the function of miR-128 in HNSCC. We established HNSCC cell lines that stably expressed individual members of the miR-128 using a lentiviral delivery system. The levels of miR-128 and their targeted proteins were analyzed by qRT-PCR and Western blot. miR-128 affinity was elucidated by MTT, colony formation, flow cytometry, and a tumor xenograft model. Two kinds of stably transfected HNSCC cell lines, including JHU-22miR-128 transfected with miR-128 and a vector control cell line JHU-22vector, were generated as a testing system. The miR-128 level in the JHU-22miR-128 cells was eleven fold higher than in JHU-22vector cells. We found that overexpression of miR-128 enable to effectively suppress cell viability (30%), decrease of proliferation (55%), and significantly reduced solid tumor growth in tumor xenografts models compared to the control groups. Transfected miR-128 cells exhibited astrocyte morphology. The inhibition mechanism of miR-128 was determined by evaluating five putative targets (BMI-1, BAG2, BAX, H3f3b, and Paip2) of miR-128. Overall, miR-128 enables to directly inhibit all these targets, but the binding affinity of miR-128 is dominantly towards to BMI-1 and BAG-2 targets. Conclusions: miR-128 exhibits anti-proliferative effects and phenotypic alteration in HNSCC by targeting multiple predicted conserved targets activity function in apoptosis, cell cycle, transcription, and translation. Our results provide a better understanding of miR-128 function in HNSCC and the potential targets to develop novel diagnostic markers and targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3170. doi:1538-7445.AM2012-3170

Abstract A13: The microRNA-200 family overcomes cisplatin resistance by targeting ceramide glycosylation

Background: Chromodomain-helicase-DNA-binding protein 5 (CHD5) is a newly identified tumor suppressor that is frequently downregulated in a variety of human cancers. Our previous work revealed that the low expression of CHD5 in colorectal cancer is correlated with CHD5 promoter CpG island hypermethylation. In this study, we investigated the effect of microRNA-211 (miR-211)-regulated CHD5 expression on colorectal tumorigenesis. Methodology/Principal Findings: miR-211 was predicted to target CHD5 by TargetScan software analysis. A stably expressing exogenous miR-211 colorectal cancer cell line (HCT-116miR-211) was generated using lentiviral transduction and used as a model for in vitro and in vivo studies. The expression level of miR-211 in HCT-116miR-211 cells was upregulated by 16-fold compared to vector control cells (HCT-116vector). Exogenous miR-211 directly binds to the 3′ untranslated region (3′ UTR) of CHD5 mRNA, resulting in a 50% decrease in CHD5 protein level in HCT-116miR-211 cells. The levels of cell proliferation, tumor growth, and cell migration of HCT-116miR-211 cells were significantly higher than HCT-116vector cells under both in vitro and in vivo conditions, as determined using the methods of MTT, colony formation, flow cytometry, tumor xenografts, and scratch assay, respectively. In addition, we found that enforced expression of miR-211 in HCT-116 cells was able to alter p53 pathway-associated regulatory proteins, such as MDM2, Bcl-2, Bcl-xL, and Bax. Conclusion/Significance: Our results demonstrate that CHD5 is a direct target of miR-211 regulation. Enforced expression of miR-211 promotes tumor cell growth at least in part by downregulating the expression level of the CHD5 tumor suppressor. Our results provide a better understanding of the association of between miR-211-regulated CHD5 expression and CHD5 function in colorectal tumorigenesis. Citation Format: Chunxiao Cai, Hassan Ashktorab, Xiaowu Pang, Yuan Zhao, Wei Sha, Yulan Liu, Xinbin Gu. The microRNA-200 family overcomes cisplatin resistance by targeting ceramide glycosylation [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A13.

Abstract 125: Exogenous MiR-125b inhibits head and neck squamous cell carcinoma growth by suppressing Bcl-2 expression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL PURPOSE: Head and neck squamous cell cancer (HNSCC) is one of top ten cancers in the United States. The overall 5-year survival rate of HNSCC has not improved in the past two decades. B-cell lymphoma 2 (Bcl-2), an oncogene, is a key regulator of the apoptosis to promote cell proliferation and apoptosis arrest. Therefore, targeting Bcl-2 is an attractive strategy for restoring a normal apoptotic process and inhibiting cancer cell growth in HNSCC. MicroRNAs (miRNAs) are a class of ∼22-nucleotide-long, endogenously expressed, highly conserved of noncoding RNAs with important regulatory functions in proliferation, apoptosis and metastasis. In this study, we screened eight cancer-associated miRNAs levels (mir-21, -124, -125b, -147, -200a, -200b, -200c and -429) in twenty five human tissue specimens and four cell lines, including twenty HNSCC specimens and five normal controls, and three HNSCC cell lines (JHU-13, -22 and -29) and one transformed oral keratinocytes cell line (OKF-6). Based on the screening data, we further investigated the mechanism of miRNA-125b (miR-125b) regulated Bcl-2 expression and the therapeutic potential of exogenous miR-125b in a miR-125b stable transfected HNSCC cell line. Design Methods: The miRNA expression level was determined by quantitative RT-PCR. A stable expressed exogenous miR-125b HNSCC cell line (JHU-22miR125b) was generated by using lentivirus transfection. Colony formation and MTT assayed cell growth and cell viability and Western blot was used to analyze protein expression. Results: The expression levels of mir-124 and mir-125b were significant lower in both tumor tissue and tumor cell lines than control, and mir-21 was significant higher in tumors. Thus, mir-125b was selected as a candidate for further study. The level of miR-125b in the transfected cell lines (JHU-22miR125b) was increased more than five times compared with the control cells (Transfected with vector only, JHU-22vector). The levels of Bcl-2 protein and cell viability were significantly lower in JHU-22miR125b than JHU-22 vector. CONCLUSION: Bcl-2 is a direct target in miR-125b regulated Bcl-2 expression. Exogenous miR-125b suppresses the proliferation and growth of cultured HNSCC cells. The effect of exogenous miR-125b on inhibition of HNSCC growth via suppressing Bcl-2 expression will be further validated in tumor xanografts. This work was supported in part by grants P20 CA118770 from National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 125. doi:10.1158/1538-7445.AM2011-125