Yc Wong

Identification of a novel function of Twist, a bHLH protein, in the development of acquired taxol resistance in human cancer cells

Taxol is one of the widely used chemotherapeutic drugs against many types of human cancer. While it is considered as one of the most effective anticancer drugs, treatment failure often occurs due to development of acquired resistance. Therefore, it is important to understand the molecular mechanisms responsible for the development of drug resistance. Although it is generally believed that taxol induces cell death through interfering with microtubules leading to mitotic arrest, recent evidence has suggested that taxol-induced cell death also occurs through pathways independent of either microtubule or mitotic arrest. In this study, we report the identification of a novel role for TWIST, a basic helix–loop–helix protein, which plays a central role in cell type determination and differentiation, during generation of acquired resistance to taxol in a nasopharyngeal carcinoma cell line, HNE1-T3, using comparative genome hybridization (CGH) and subsequent RT–PCR and Western blotting. We found that upregulation of TWIST was associated with cellular resistance to taxol but not other drugs with different mechanisms of action. The fact that increased TWIST protein levels were also associated with another microtubule-targeting anticancer drug, vincristine, in four types of human cancer including nasopharyngeal, bladder, ovarian and prostate, indicates that it may play a central role in the resistance to microtubule-disrupting agents. In addition, ectopic expression of TWIST into human cancer cells also led to increased resistance to both taxol and vincristine. Our results indicate a novel mechanism that leads to resistance to microtubule-disrupting anticancer drugs through upregulation of TWIST. Our evidence provides a therapeutic strategy to overcome acquired resistance through inactivation of TWIST expression in human cancer.

FTY720, a fungus metabolite, inhibits in vivo growth of androgen‐independent prostate cancer

FTY720, a derivative of fungus, has demonstrated dramatic anticancer effect in several malignancies recently. Our study evaluates the therapeutic potential of FTY720 in the treatment of androgen‐independent prostate cancer using a human prostate cancer xenograft in nude mice. CWR22R, an androgen‐independent human prostate tumor xenograft was inoculated into castrated nude mice and the animals were administrated with either normal saline or FTY720 (10 mg/kg) through intraperitoneal (i.p.) injection for 20 days. Body weight and tumor volume were recorded every 2 days, and serum prostate specific antigen (PSA) levels were also measured before and after the treatment. The effect of FTY720 on tumor cell proliferation was examined using antibodies against PCNA and Ki‐67 by immunohistochemical staining, MTT assay and colony forming assay, whereas apoptotic effect of FTY720 was evaluated by TUNEL assay and immunostaining using antibodies against cleaved caspase 3 and Bcl‐2. In addition, the potential inhibitory effect of FTY720 on prostate cancer angiogenesis and metastasis was investigated by immunostaining of CD31, VEGF, E‐cadherin and β‐catenin. Our results showed that FTY720 treatment led to suppression of CWR22R tumor growth without causing any detectable side effects in nude mice. The FTY720‐induced tumor suppression was correlated with decreased serum PSA level as well as reduced proliferation rate, suppression of angiogenic factors, and restoration of E‐cadherin and β‐catenin expression. In addition, the FTY720‐treated tumors showed increased apoptosis rate demonstrated by increased TUNEL‐ and cleaved caspase 3‐positive cells, and decreased Bcl‐2 expression. Our results suggest a potential novel agent in the suppression of androgen‐independent prostate cancer.

Inactivation of Human MAD2B in Nasopharyngeal Carcinoma Cells Leads to Chemosensitization to DNA-Damaging Agents

Rev7p has been suggested to play an important role in regulating DNA damage response in yeast, and recently, the human homologue (i.e., MAD2B) has been identified, which shares significant homology to the mitotic checkpoint protein MAD2. In this study, we investigated whether MAD2B played a key role in cellular sensitivity to DNA-damaging anticancer drugs by suppressing its expression using RNA interference in nasopharyngeal carcinoma cells. Using colony formation assay, we found that suppression of MAD2B conferred hypersensitivity to a range of DNA-damaging agents, especially DNA cross-linkers, such as cisplatin, and gamma-irradiation. This effect was associated with reduced frequencies of spontaneous and drug-induced mutations, elevated phosphorylation of histone H2AX, and markedly increased chromosomal aberrations in response to DNA damage. In addition, there was also a significant decrease in cisplatin-induced sister chromatid exchange rate, a marker for homologous recombination-mediated post-replication repair in MAD2B-depleted cells. These results indicate that MAD2B may be a key factor in regulating cellular response to DNA damage in cancer cells. Our findings reveal a novel strategy for cancer therapy, in which cancer cells are sensitized to DNA-damaging anticancer drugs through inactivation of the MAD2B gene.

Epigenetic inactivation of CHFR in nasopharyngeal carcinoma through promoter methylation

Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers. Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. Epigenetic inactivation of CHFR through promoter CpG hypermethylation may lead to CIN and has been reported in several human cancers. In this study, we investigated the CHFR gene expression in a panel of nasopharyngeal carcinoma (NPC), prostate, ovarian, and breast cancer cell lines. We found that the expression of CHFR mRNA was significantly decreased or undetectable in all eight NPC cell lines as well as three human NPC xenografts, whereas non‐malignant nasopharyngeal cell lines and other cancer cell lines tested expressed CHFR at relatively high levels. Hypermethylation of CHFR promoter region was also strongly correlated with decreased CHFR expression in NPC cell lines and xenografts. Treatment with a methyltransferase inhibitor, 5‐aza‐2′‐deoxycytidine, led to restoration of CHFR expression in NPC cell lines. More importantly, hypermethylation of CHFR promoter region was detected in 61.1% (22 out of 36) of primary NPC tumors while it was absent in non‐malignant tissues. These findings suggest that downregulation of CHFR is a common event in NPC cells which may be due to hypermethylation of the gene promoter region.

Id proteins expression in prostate cancer : high-level expression of Id-4 in primary prostate cancer is associated with development of metastases

A major cause of treatment failure for prostate cancer is the development of androgen-independent metastatic disease. Id protein family, a group of basic helix–loop–helix transcription factors, has been shown to be involved in carcinogenesis and a prognostic marker in several types of human cancers. In this study, we examined the expressions of four Id proteins, Id-1, -2, -3 and -4, in 125 clinical prostate cancer specimens as well as 40 nodular hyperplasia specimens by immunohistochemistry. The expressions of Id proteins were correlated with Gleason grading and metastatic progress of the tumors. We found that Id proteins were dysregulated in prostate cancer. Id-1 and -2 expressions were elevated while Id-3 and -4 expressions were reduced in prostate cancers compared to nodular hyperplasia. Cytoplasmic staining of Id-1 (P=0.013) and nuclear staining of Id-2 (P=0.001) and Id-4 (P<0.001) were positively correlated with Gleason score. The results indicate that these Id proteins may play a positive role in the development of prostate cancer. In contrast, Id-3 might have an inverse relationship with prostate neoplastic transformation (P=0.002) and cancer progression (P=0.022). We found that Id-4 nuclear overexpression in the primary prostate cancers significantly increased the risks to the development of metastasis in the patients (odds ratio=3.215, 95% confidence interval=1.150–8.987, P=0.026). Our results suggest that in prostate cancer patients, differential Id proteins expressions may be a useful marker for poor prognosis, and Id-4 may be a potential prognostic marker for distant metastasis.

Inactivation of Id‐1 in prostate cancer cells: A potential therapeutic target in inducing chemosensitization to taxol through activation of JNK pathway

Resistance to anticancer drugs is the major problem in the treatment of many advanced cancers, including androgen‐independent prostate cancer. Recently, increased expression of Id‐1, a basic helix‐loop‐helix protein, is reported in several types of advanced cancer. It is suggested that high expression of Id‐1 may provide an advantage for cancer cell survival and inactivation of Id‐1 may be able to increase cancer cells susceptibility to apoptosis. To test this hypothesis, in this study, by using RNA interfering technology, we inactivated the Id‐1 gene in 2 androgen‐independent prostate cancer cell lines, DU145 and PC3, and investigated whether downregulation of Id‐1 could lead to increased sensitivity to a commonly used anticancer drug, taxol. By using colony forming assay and MTT assay, we found that inactivation of Id‐1 resulted in both decreased colony forming ability and cell viability in prostate cancer cells, after taxol treatment. In addition, the si‐Id‐1‐induced sensitization to taxol was associated with activation of apoptosis pathway, which is demonstrated by increased apoptotic index, DNA laddering, sub‐G1 phase of the cell cycle, as well as cleaved‐PARP and Caspase 3. Furthermore, c‐Jun N‐terminal kinase (JNK), one of the common pathways responsible for taxol‐induced apoptosis, was also activated in the si‐Id‐1 transfected cells. Inhibition of JNK activity by a specific inhibitor, SP600125, blocked the si‐Id‐1‐induced sensitivity to taxol. These results indicate that increased Id‐1 expression in prostate cancer cells may play a protective role against apoptosis, and downregulation of Id‐1 may be a potential target to increase sensitivity of taxol‐induced apoptosis in prostate cancer cells.

Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to apoptotic stress.

Epstein–Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV‐encoded RNAs (EBERs) are the most abundant EBV transcripts (about 107 copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double‐stranded RNA (dsRNA) and may bind to the double‐stranded RNA‐dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER‐expressing plasmid, pESK10. The EBER‐expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER‐expressing NP69 cells did not form colonies in soft agar and were non‐tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER‐expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl‐2 were observed in EBER‐expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase‐3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER‐expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen‐activated protein kinase) and c‐jun were also observed in EBER‐expressing NP cells. Our results suggest that EBER expression may confer an apoptotic‐resistant phenotype in immortalized nasopharyngeal epithelial cells.

Mitotic checkpoint: A therapeutic target in the treatment of human cancer

The most common feature of human cancer cells is aneuploidy and a defective mitotic checkpoint is thought to be responsible. Since a group of anticancer drugs target the mitotic checkpoint, this review will discuss the association of mitotic checkpoint with chemotherapeutic drug sensitivity and suggest a novel therapeutic tool to achieve chemosensitization in human cancer cells.