Tsun Yee Tsang

p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3

Development of drug resistance is one of the major obstacles in cancer chemotherapy. The molecular mechanism leading to drug resistance is still not fully understood. A10A cells, a doxorubicin-resistant subline of human squamous cell carcinoma A431 cells, showed cross-resistance to methotrexate and also resistance to the drug-induced apoptosis. The cells also showed overexpression of a mutated form of p53, p53-R273H (Arg to His at codon 273), and down-regulation of procaspase-3. Knockdown of p53-R273H by p53 small interfering RNA in A431 cells increased procaspase-3 level and sensitized the cells to drug-induced apoptosis. On the other hand, transfection of p53-R273H into p53 null human osteosarcoma Saos-2 cells down-regulated procaspase-3 level and induced resistance to the drug toxicity and drug-induced apoptosis. The results support the idea that p53-R273H may gain new functions in induction of drug resistance and impairment in drug-induced apoptosis through down-regulation of procaspase-3 level. The study sheds new light on the understanding of the gain of function and drug resistance mechanisms associated with mutant p53. [Mol Cancer Ther 2007;6(3):1054–8]

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5′-serial truncation analysis of the MDR1 promoter defined a region from position −450 to −193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.

Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells

Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.

P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells.

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.

Mechanistic Study on Growth Suppression and Apoptosis Induction by Targeting Hepatoma-derived Growth Factor in Human Hepatocellular Carcinoma HepG2 Cells

Hepatoma-derived growth factor (HDGF) is frequently overexpressed in human cancer. The growth factor was previously demonstrated to be a survival factor as knock-down of HDGF suppresses the growth and induces apoptosis in human cancer cells through the Bad-mediated intrinsic apoptotic pathway. However, inactivation of Bad cannot completely repress the apoptosis induced upon HDGF knock-down, indicating the presence of other unidentified pathways. In the present study, HDGF knock-down was shown to trigger the Fas-mediated extrinsic apoptotic pathway in human hepatocellular carcinoma HepG2 cells through NF-κB signaling pathway. Increases in Fas expression and fas promoter activity were detected upon HDGF knock-down by Western blot analysis and luciferase reporter assay. Knock-down of fas inhibited HDGF knock-down effect on apoptosis induction and growth suppression as revealed by annexin V binding assay and soft agar assay. Down-regulation of IκBα was also observed upon HDGF knock-down. Overexpression of IκBα by transient transfection or inhibition of NF-κB by BAY11-7082 suppressed HDGF knock-down effect on fas promoter activation, Fas up-regulation, apoptosis induction and growth suppression. Furthermore, the interaction of Fas-mediated extrinsic and Bad-mediated intrinsic apoptotic pathways was demonstrated as a stronger inhibition on apoptosis induction and growth suppression upon HDGF knock-down was observed when both pathways were inactivated. The results therefore suggested that, through both intrinsic and extrinsic apoptotic pathways, HDGF may function as a survival factor and be a potential target for cancer therapy.

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway.

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, γ-radiation, IFN-α, and IFN-γ in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance. [Mol Cancer Ther 2008;7(10):3160–8]

Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3

Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.

Abstract 199: HPV-16 E6 silencing of p16INK4a in human cervical cancer cells

Aberrant promoter methylation and silencing of tumor suppressor genes such as p16INK4a were reported in association with cervical carcinogenesis. Persistent infection with high-risk human papillomavirus (HPVs), such as HPV-16, contributes significantly towards the cervical cancer development. One of the HPV-16 early proteins E6 is considered to function as an oncoprotein. In the present study, the role of HPV-16 E6 in the regulation of p16INK4a silencing was investigated. Knockdown of E6 in HPV-16 positive human cervical carcinoma SiHa cells by the technique of small interfering RNA (siRNA) led to an increase in p16INK4a mRNA expression as assessed by quantitative RT-PCR analysis. Addition of DNA demethylating agent, 5-aza-2’-deoxyctidine, also had similar effect. Moreover, by methylation-specific PCR analysis, hypo-methylation of p16INK4a promoter was detected in HPV-16 E6 knockdown SiHa cells. The results indicated that HPV-16 E6 may regulate the expression of p16INK4a through promoter methylation. By Western blot analysis, the expression of DNA methyltransferase DNMT1 was repressed in HPV-16 E6 knockdown SiHa cells while it was induced in HPV-16 E6 over-expressed cells. In addition, decrease in p16INK4a mRNA expression and hyper-methylation of its promoter were observed in SiHa cells upon DNMT1 cDNA transfection. To sum up, HPV-16 E6 silencing of p16INK4a may be through the upregulation of DNMT1 which in turn induces the hyper-methylation of p16INK4a promoter and thus suppresses p16INK4a expression. This pathway may be one of the possible mechanisms for the oncogenic function of HPV-16 E6 in human cervical cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 199.

Abstract 1702: S-adenosyl-L-methionine induces P-glycoprotein overexpression and drug resistance in human cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL P-glycoprotein (Pgp) overexpression is frequently associated with multidrug resistance in cancer cells. Pgp is an efflux pump and is encoded by human mdr1 gene. The expression of mdr1 may be regulated by promoter methylation catalyzed by DNA methyltransferases (DNMTs). S-adenosyl-L-methionine (SAMe) is an important cofactor of DNMTs. Although SAMe has been shown to regulate promoter methylation of various genes, the effect of SAMe on Pgp expression and chemosensitivity of cancer cells are however not known. The aim of the present study is to examine the role of SAMe in the regulation of Pgp expression and drug sensitivity of human cancer cells. By qRT-PCR and Western blot analyses, incubation with 1mg/ml SAMe for 24h was found to increase mdr1 mRNA and Pgp protein levels in human hepatocellular carcinoma HepG2 cells, human colorectal adenocarcinoma CaCO-2 cells and human leukemia Jurkat-T cells. Methylation specific PCR and bisulfite DNA sequencing analyses revealed that the SAMe treatment induces hypomethylation of mdr1 promoter. Moreover, SAMe decreased intracellular doxorubicin accumulation and increased doxorubicin resistance in human cancer cells as assessed by doxorubicin accumulation assay and MTT assay. Furthermore, in human colorectal carcinoma HCT-116 parent cells and DNMTs knock-out sublines, SAMe induced Pgp expression in HCT-116 parent and DNMT3b knock-out subline but not in DNMT1 and DNMT1/DNMT3b double knockout sublines. The results from the present study therefore suggest that SAMe induces hypomethylation of mdr1 promoter, Pgp expression, and thus drug resistance in human cancer cells. Furthermore, DNMT1 appeared to be important in the effect of SAMe on mdr1. 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 1702. doi:10.1158/1538-7445.AM2011-1702

Abstract 3960: MicroRNA-23b targeting urokinase-type plasminogen activator facilitates human papillomavirus type 16 E6 induction of cervical cancer cell migration

Aberrant expression of microRNA in human cervical cancer is associated frequently with human papillomavirus (HPV) integration. MicroRNA-23b (miR-23b) is often down-regulated in HPV-associated cervical cancer. Also, urokinase-type plasminogen activator (uPA), a target of miR-23b, is reported to be over-expressed in cervical cancer. Thus, in the present study, the functional role of miR-23b in HPV-16 E6 associated cervical cancer development was investigated. Knockdown of oncoprotein E6 by E6 siRNA in HPV-16 positive human cervical carcinoma SiHa cells and CaSki cells led to an increase in the expression of miR-23b and a decrease in the expression of uPA as assessed by quantitative RT-PCR and Western blot analyses. Besides, uPA is confirmed to be the target of miR-23b as transient transfection of SiHa cells with miR-23b precursor vector repressed uPA expression and also the activity of luciferase reporter carrying the 3’UTR of uPA mRNA. uPA is known to be involved in cell migration and cell invasion. By wound healing assay, the cell migration rate of SiHa cells was reduced upon E6 as well as uPA knockdown. Moreover, the effect of E6 siRNA on cell migration was offset upon transfection with anti-miR-23b inhibitor. The results indicate that HPV-16 E6 may regulate cell migration through miR-23b/uPA pathway. Furthermore, p53 is a well-known gene target of HPV-16 E6. Knockdown of p53 by p53 siRNA decreased the miR-23b expression and increased the uPA expression. This shows that p53 may mediate the E6 effect on miR-23b/uPA expressions and thus cell migration of human cervical cancer. 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 3960. doi:10.1158/1538-7445.AM2011-3960